Constitutive Yeast LLP Promotor-Based Expression Systems

ABSTRACT

The present invention belongs to the field of biotechnology, recombinant protein production, molecular biology, microbiology and microbial genetics. It provides a modified eukaryotic cell that is modified to the effect that the modified eukaryotic cell is not able to provide an SSN6-like related protein or an SSN6-like protein that exerts its wildtype function and/or wildtype activity, the amount of SSN6-like related protein or of SSN6-like protein being present in the modified eukaryotic cell differs from the amount of SSN6-like related protein or of SSN6-like protein being present in its wildtype form, and/or essentially no SSN6-like related protein or SSN6-like protein is present in the modified cell. Additionally, the present invention provides a polynucleotide sequence comprising a modified ssn6-like related gene or modified ssn6-like gene, and a vector comprising said polynucleoptide. Additionally provided is an expression vector comprising a promoter that is repressed in the presence of SSN6-like protein or SSN6-like related protein, and a host cell comprising said vectors. The present invention further refers to a method for determining the purity of a composition by using the modified eukaryotic cell, to a method of expressing gene(s) of interest, and eukaryotic cells comprising modified ssn6-like related gene or ssn6-like gene.

FIELD OF THE INVENTION

The present invention belongs to the field of biotechnology, recombinantprotein production, molecular biology, microbiology and microbialgenetics. Specifically, the present invention relates to a modifiedeukaryotic cell, or expression system, respectively, comprising amodified gene, a polynucleotide sequence comprising said modified gene,an expression vector comprising said polynucleotide sequence, a hostcell comprising said expression vector, as well as to the use of saidcomponents.

Further, the present invention refers to methods using said components.

DESCRIPTION OF THE BACKGROUND ART

Expression systems for the expression of genes of interest (GOI) andalso for the production of proteins of interest are widely known and ofgreat importance in the field of molecular biology, microbiology andmicrobial genetics.

Until now, there are several expression systems known that allow theexpression of certain desired genes and proteins, such as cell-basedsystems and cell-free systems. Cell-based systems comprise bacterialsystems and eukaryotic systems, and cell-free systems are based forinstance on the use of purified RNA polymerase, ribosomes, tRNA andribonucleotides or are based on the use of systems containing cell-freeprotein synthesis systems such as for example rabbit reticulocytelysates. The choice of the respectively used expression system dependson various factors such as the type of protein (eukaryotic orprokaryotic source of the protein) that is to be expressed, or whetherpost-translational modifications are necessary in order to ensure properfunction and/or structure.

For instance, in general, if eukaryotic proteins are to be expressed,typically a eukaryotic expression system is selected. The reasontherefore is that most eukaryotic proteins are modified for instance byphospohorylation, acylation, methylation, ubiquitylation orglycosylation. Bacteria are known to be no ideal host organisms for suchmodified proteins, as bacteria are prokaryotes which are not equippedwith the full enzymatic machinery to accomplish the post-translationalmodifications or molecular folding that are required for many proteinsto function properly. Thus, if eukaryotic proteins are to be expressed,typically expression systems using eukaryotic cells are used. Suchexpression systems are for instance based on yeast, fungi or insectcells. Expression systems in yeast use either inducible or non-induciblepromoters for expression. Inducible promoters for methylotrophic yeastsuch as Pichia pastoris (P. Pastoris) often require the use of methanolas inducing substance.

Despite the known expression systems, there is still a need for animproved expression system, in particular for a eukaryotic expressionsystem. Improvements are for instance desired with regard to thehandling of the expression system, costs, safety aspects (e.g. avoidingthe use of highly inflammable inducers such as methanol), complexity ofthe system, the time needed for expressing the proteins, or the yield ofthe expressed proteins.

SUMMARY OF THE INVENTION

The present invention provides the following aspects, subject-mattersand preferred embodiments, which respectively taken alone or incombination, contribute to solving the object of the present invention:

(1) A modified eukaryotic cell, which is modified compared to itswildtype cell at least in that it comprises

-   -   a modified ssn6-like gene or a modified ssn6-like related gene,        and/or    -   a modified expression level of SSN6-like protein or of SSN6-like        related protein, or    -   in that    -   an ssn6-like gene or an ssn6-like related gene is deleted,

respectively to the effect that

-   -   the modified eukaryotic cell is not able to provide an SSN6-like        protein or an SSN6-like related protein that exerts its wildtype        function and/or wildtype activity,    -   the amount of SSN6-like protein or of SSN6-like related protein        being present in the modified eukaryotic cell differs from the        amount of SSN6-like protein or of SSN6-like related protein        being present in its wildtype form, and/or    -   no SSN6-like protein or SSN6-like related protein is present in        the modified cell,

wherein said modified eukaryotic cell, compared to its wildtype cell,exhibits different SSN6-like protein or SSN6-like related-proteinactivity and/or function, preferably with respect to the proteins'ability in regulating gene expression.

In a preferred embodiment, said modified eukaryotic cell exhibitsdifferent SSN6-like protein or SSN6-like related protein activity and/orfunction with respect to the proteins' activity and/or function inregulating the expression of genes that are under control of thelectin-like protein (LLP) promoter.

(2) The modified eukaryotic cell according to item (1), wherein saidmodified eukaryotic cell exhibits reduced SSN6-like protein or SSN6-likerelated-protein activity and/or reduced function, or no SSN6-likeprotein or SSN6-like related-protein activity and/or function at all,preferably with regard to the proteins' activity and/or function inregulating gene expression, preferably in regulating the expression ofgenes that are under control of the LLP promoter.

(3) The modified eukaryotic cell according to item (1) or (2), whereinthe ssn6-like gene and/or the ssn6-like related gene is/are modified ina regulatory sequence, such as promoter(s), enhancer(s), terminator(s),silencer(s), IRES-sequence(s), ribosome-binding site(s), and sequence(s)stabilizing or destabilizing the mRNA by secondary structure(s), and/orin a coding sequence.

(4) The modified eukryotic cell according to any of items (1) to (3),wherein the modified expression level of SSN6-like protein or ofSSN6-like related protein is obtained by modifying the coding sequenceof the ssn6-like gene and/or of the ssn6-like related gene.

(5) The modified eukaryotic cell according to any of items (1) to (4),wherein the total activity, function, preferably gene regulatoryfunction, and/or amount of SSN6-like protein or SSN6-like relatedprotein of the modified eukaryotic cell is at most 20%, preferably atmost 15%, more preferably at most 10%, even more preferably at most 5%of the total activity, function and/or amount of SSN6-like protein orSSN6-like related protein of its wildtype form, and most preferablythere is no activity and/or no function of the SSN6-like protein orSSN6-like related protein of the modified eukaryotic cell at all.

(6) The modified eukaryotic cell according to any of items (1) to (5),wherein the ssn6-like gene or the ssn6-like related gene and/or theexpression level of the SSN6-like protein or of the SSN6-like relatedprotein is modified by

-   -   introduction of one or more point mutations, e.g. substitution,        insertion or deletion of a single or more nucleotides in the        polynucleotide sequence of the ssn6-like gene or of the        ssn6-like related gene,    -   partial or complete deletion of the polynucleotide sequence of        the ssn6-like gene or of the ssn6-like related gene, and/or    -   partial or complete replacement of the polynucleotide sequence        of the ssn6-like gene or of the ssn6-like related gene by a        different, e.g. heterologous, nucleotide sequence,

wherein the polynucleotide sequence of the ssn6-like gene or of thessn6-like related gene comprises coding and regulatory polynucleotidesequences, and wherein said regulatory polynucleotide sequences of thessn6-like gene or of the ssn6-like related gene comprise promoters,enhancers, terminators, silencers, IRES-sequences, ribosome-bindingsites, or sequences stabilizing or destabilizing the mRNA by secondarystructures.

(7) The modified eukaryotic cell according to any of the precedingitems, wherein said cell is a fungal cell, preferably a yeast cell, morepreferably selected from the group consisting of Saccharomyces species(e.g., Saccharomyces cerevisiae), Kluyveromyces species (e.g.,Kluyveromyces lactis), Torulaspora species, Yarrowia species (e.g.,Yarrowia lipolitica), Schizosaccharomyces species (e.g.,Schizosaccharomyces pombe), Pichia species (e.g., Pichia pastoris orPichia methanolica), Hansenula species (e.g., Hansenula polymorpha),Torulopsis species, Komagataella species, Candida species (e.g., Candidaboidinii), and Karwinskia species, even more preferably the eukaryoticcell is a Pichia cell, most preferably a Pichia pastoris cell accordingto current classification.

(8) The modified eukaryotic cell according to item (7), wherein the cellcan be any Pichia pastoris cell, preferably a cell of a strain selectedfrom the group consisting of NRRL Y-11430, CBS704, GS115, and KM71.

(9) The modified eukaryotic cell according to any of the precedingitems, wherein said modified eukaryotic cell comprises a polynucleotidesequence which represents a modification of SEQ ID NO: 1, preferably themodified eukaryotic cell comprises SEQ ID NO: 7.

SEQ ID NO: 1 represents the coding region of the ssn6-like gene.

SEQ ID NO: 7 represents modified ssn6-like nucleotide sequence.

(10) The modified eukaryotic cell according to any of the proceedingitems, wherein said wildtype cell contains a SSN6-like or SSN6-likerelated protein, which protein comprises one or both of the consensusamino acid sequences depicted in SEQ ID NO: 63 and 64.

In other words, the eukaryotic cell, prior to its modification resultingin said modified eukaryotic cell, contained an SSN6-like or SSN6-likerelated protein, which protein comprised one or both of the amino acidsequences depicted in SEQ ID NO: 63 and 64.

(11) A polynucleotide sequence comprising, preferably consisting of, amodified ssn6-like gene or nucleotide sequence, e.g. as depicted in SEQID NO: 7, or a modified ssn6-like related gene,

wherein, if said polynucleotide sequence

(a) is introduced into a suitable expression system, and tried to beexpressed, essentially no SSN6-like protein or SSN6-like related proteinis expressed, or

(b) is expressed in a suitable expression system, SSN6-like protein orSSN6-like related protein is expressed that does not exert its wildtypefunction and/or wildtype activity, preferably with respect to itsactivity and/or function in regulating gene expression, more preferablyin regulating the expression of genes that are under control of the LLPpromoter, and/or

(c) is expressed in a suitable expression system, an amount of SSN6-likeprotein or SSN6-like related protein is expressed that differs from theamount of wildtype SSN6-like protein or of SSN6-like related protein.

In order to assess any of (a) to (c), the corresponding wildtypepolynucleotide sequence is tried to be expressed under comparable or thesame conditions, under which the modified ssn6-like gene or modifiedssn6-like related gene is tried to be expressed.

(12) The polynucleotide sequence according to item (11), wherein, ifsaid polynucleotide sequence is introduced into a suitable expressionsystem, and tried to be expressed, or is expressed in a suitableexpression system, the amount of SSN6-like protein or SSN6-like relatedprotein is reduced, or there is essentially no, preferably no, SSN6-likeprotein or SSN6-like related protein at all present, compared to theamount of SSN6-like protein or SSN6-like related protein that isexpressed when the corresponding wildtype polynucleotide sequence isexpressed under comparable or the same conditions.

(13) The polynucleotide sequence according to item (11) or (12), whereina regulatory sequence of the ssn6-like gene or of the ssn6-like relatedgene is selected from the group comprising promoters, enhancers,terminators, silencers, IRES-sequences, ribosome-binding sites, andsequences stabilizing or destabilizing the mRNA by secondary structures,preferably said regulatory sequence is selected from the groupconsisting of promoters, enhancers, and terminators.

(14) The polynucleotide sequence according to any of items (11) to (13),wherein the ssn6-like gene or the ssn6-like related gene has beenmodified as defined in item (6).

(15) The polynucleotide sequence according to any of items (11) to (14),wherein the polynucleotide sequence comprises, preferably consists of,modifications of SEQ ID NO: 1, preferably the polynucleotide sequencecomprises, more preferably consists of, SEQ ID NO: 7.

(16) A nucleic acid sequence comprising the polynucleotide sequenceaccording to any of items (11) to (15).

(17) A vector comprising the polynucleotide sequence/nucleic acidsequence according to any of items (11) to (16).

(18) A host cell comprising the vector according to item (17), or apolynucleotide according to any of items (11) to (13).

(19) The host cell according to item (18), wherein said host cell is abacterium, preferably Escherichia coli.

(20) A polypeptide encoded by the polynucleotide sequence according toany of items (11) to (15).

(21) An expression vector comprising a promoter, wherein said promoteris characterized in that it is repressed in the presence of SSN6-likerelated protein or SSN6-like protein if said expression vector isintroduced into a suitable expression system, e.g. into a eukaryoticcell as defined in item (7) or (8).

(22) The expression vector according to item (21), which furthercomprises one or more gene(s) of interest.

(23) The expression vector according to item (22), wherein the gene(s)of interest is/are under control of the promoter defined in item (21).

(24) The expression vector according to any of items (21) to (23),wherein said promoter is an LLP promoter, preferably an LLP promotercomprising, preferably consisting of, SEQ ID NO: 2 or 12, or consistingof a sequence having a length of 1000, 775, 675, 605, 576, 512, 472,415, 404, 372, 305, 285, 235, 165, or 100 nucleotides counted in eachcase from the 3′-end of SEQ ID NO: 12, or modified versions thereof,said modified versions being characterized in that they still exhibitthe promoter function.

(25) The expression vector according to any of items (21) to (24),wherein, if the promoter is an LLP promoter as defined in item (24), LLPprotein is not encoded by the polynucleotide sequence of this expressionvector, and preferably the gene of interest is selected from the groupconsisting of genes encoding enzymes, antibodies or fragments thereof,hormones, structural proteins (such as albumin), and protein-antigensbeing suitable for vaccines.

(26) The expression vector according to any of items (21) to (24),wherein, if the promoter is an LLP promoter as defined in item (24), LLPprotein is encoded by the polynucleotide sequence of this expressionvector, and preferably the gene of interest is selected from the groupconsisting of genes encoding enzymes, antibodies or fragments thereof,hormones, structural proteins (such as albumin), and protein-antigensbeing suitable for vaccines.

(27) The expression vector according to any of items (21) to (26),wherein said expression vector further comprises features selected fromany one of the following:

-   -   (i) a selection marker;    -   (ii) a purification marker;    -   (iii) a signal sequence, preferably an alpha-factor secreting        signal sequence, more preferably the MFalpha pre-pro signal        sequence, even more preferably a signal sequence comprising or        consisting of, preferably consisting of, SEQ ID NO: 14 or SEQ ID        NO: 21;    -   (iv) an origin of replication; and/or    -   (v) a nucleotide sequence for targeted and/or random integration        into the genome of a host cell.

In a preferred embodiment, the signal sequence (iii) of the expressionvector according to item (27) comprises or consists, preferably consistsof, SEQ ID NO: 14.

(28) The expression vector according to any of items (21) to (27),wherein said vector contains more than one promoter as defined in item(21), and/or wherein said vector contains more than one LLP promotoraccording to item (24), preferably 2, 3, 4, 5, or 6 LLP promotersaccording to item (24) and preferably different LLP promoters, e.g.different length LLP-promoters, according to item (24) which result indifferent expression rates of the genes of interest under control ofsaid LLP promoter.

(29) The expression vector according to any of items (21) to (27),wherein said vector contains, besides one or more LLP promoters, inaddition one or more other promoters different from a LLP promoter,which other promoters result in expression rates different to theexpression rates of the LLP promoters.

(30) Use of an expression vector according to item (28) or (29) for theexpression of a multimeric protein, said multimeric protein consistingof two or more individual protein chains, which individual proteinchains are connected to each other by one or more disulfide bridgesand/or which individual protein chains form a multimeric protein byother forms of protein chain-protein chain interactions.

(31) Use according to item (30) wherein said multimeric protein isexpressed by transforming a cell with two or more individual vectors,wherein each vector is containing one or more promoter(s) as defined initem (21) and/or in item (24), or wherein at least one vector contains aLLP promoter and at least one vector contains other promoter differentfrom a LLP promoter.

(32) A host cell, comprising the vector according to item (17) or any ofitems (21) to (29).

(33) The host cell according to item (32), wherein said host cell is abacterium, preferably Escherichia coli, or the modified eukaryotic cellaccording to any of items (1) to (9).

(34) The modified eukaryotic cell according to any of items (1) to (10),comprising the expression vector according to any of items (21) to (29).

(35) An expression system, comprising

-   -   a) the modified eukaryotic cell as defined in any of items (1)        to (10);    -   b) the expression vector as defined in any of items (21) to        (29), wherein said expression vector can also be present in        linearized form and/or at least parts of the vector being        integrated into the genome of the modified eukaryotic cell.

(36) The modified eukaryotic cell according to any of items (1) to (10),further comprising a promoter as defined in item (21) or (24).

(37) The modified eukaryotic cell according to item (36), furthercomprising (a) gene(s) of interest being under control of the promoteras defined in item (21) or (24).

(38) The modified eukaryotic cell according to item (37), wherein thegene(s) of interest is (are) as defined in item (25).

(39) The modified eukaryotic cell according to item (37) or (38),wherein the LLP gene is not expressed in addition to the gene(s) ofinterest, or wherein the LLP gene is expressed in addition to thegene(s) of interest.

(40) The modified eukaryotic cell according to item (39),

-   -   wherein the LLP promoter or the LLP-like promoter controls (a)        gene(s) of interest which is (are) different from LLP, and in        addition another copy of said promoter controls the LLP-gene        (e.g. Option 2 in FIG. 3D); or    -   wherein the LLP promoter or the LLP-like promoter only        controls (a) gene(s) of interest which is (are) different from        LLP and no LLP-gene is present (e.g. Option 1 in FIG. 3C).

(41) Use of

-   -   (A) the modified eukaryotic cell,    -   (B) the polynucleotide sequence,    -   (C) the expression vector,    -   (D) the host cell, or    -   (E) an expression system

according to any of items (1) to (40)

in a method of expressing, preferably overexpressing, gene(s) ofinterest.

(42) Method for determining the purity of a composition comprising theexpression product of a gene of interest, comprising the followingsteps:

-   -   (a) expressing gene(s) of interest by using a modified        eukaryotic cell according to any of items (1) to (10) and by        using an expression vector according to item (25), wherein        -   (a1) the modified eukaryotic cell comprises a gene encoding            LLP protein under control of an LLP promoter or an LLP-like            promoter, and wherein        -   (a2) the expression vector comprises one or more gene(s) of            interest under control of an LLP promoter or an LLP-like            promoter, wherein said gene(s) of interest does (do) not            encode LLP,    -   thereby obtaining a composition comprising the expression        product of the gene(s) of interest, i.e. the protein(s) of        interest, and the LLP protein;    -   (b) determining the amount of the expression product of the        gene(s) of interest, i.e. the amount of the protein(s) of        interest, and the amount of LLP protein being present in the        composition obtained in step (a), wherein the amount of LLP        protein compared to the amount of expression product of the        gene(s) of interest, i.e. of the protein(s) of interest, is        indicative for the purity of the composition obtained in step        (a); and, optionally,    -   (c) subjecting the composition of step (a) to one or more        downstream purification step(s), followed by step (b) for        determining the amount of the the protein(s) of interest, and        the amount of LLP protein being present in the composition        obtained after having carried out said downstream purification        step, i.e. monitoring host-cell-protein depletion (purity of        gene of interest protein in the course of its purification).

(43) A method of expressing one or more gene(s) of interest in aeukaryotic cell comprising an ssn6-like gene or an ssn6-like relatedgene, wherein the translation of the mRNA transcript of the ssn6-likegene or the ssn6-like related gene is prevented by hybridizing acomplementary sequence or a partial sequence thereof to the mRNAtranscript.

(44) The method of item (43), wherein the partial sequence of thecomplementary sequence is a siRNA, anti-sense RNA, a ribozyme, ortriplex RNA or DNA.

(45) A method of expressing one or more gene(s) of interest in aeukaryotic cell comprising an SSN6-like related expression cassette orSSN6-like expression cassette, wherein the SSN6-like related protein orthe SSN6-like protein in said eukaryotic cell is modified or inhibitedin its function and/or activity.

(46) The method according to item (45), wherein the SSN6-like relatedprotein or SSN6-like protein of said eukaryotic cell is modified orinhibited in its function and/or activity in regulating the LLPpromoter, preferably the SSN6-like related protein or SSN6-like proteinexhibits reduced SSN6-like protein or SSN6-like related-protein activityand/or function, or no SSN6-like protein or SSN6-like related-proteinactivity and/or function at all.

(47) A eukaryotic cell comprising

-   -   a modified ssn6-like gene or a modified ssn6-like related gene,        wherein said gene has inserted a foreign nucleotide sequence, to        the effect that    -   (i) the eukaryotic cell is not able to provide an SSN6-like        protein or an SSN6-like related protein that exerts its wildtype        function and/or wildtype activity, preferably with respect to        its activity and/or function in regulating the LLP promoter or        LLP-like promoter,    -   (ii) the amount of SSN6-like protein or of SSN6-like related        protein being present in the eukaryotic cell differs from the        amount of SSN6-like protein or of SSN6-like related protein        being present in its wildtype form, preferably the amount of the        SSN6-like protein or of the SSN6-like related protein is        reduced, and/or    -   (iii) no SSN6-like protein or SSN6-like related protein is        present in the eukaryotic cell,

and

-   -   a gene of interest that replaces a part or all of the coding        region of the gene encoding the LLP protein, and that is under        control of the LLP promoter, to the effect that essentially no        LLP protein is present in the eukaryotic cell,

preferably wherein said eukaryotic cell exhibits reduced SSN6-likeprotein or SSN6-like related-protein activity and/or function, or noSSN6-like protein or SSN6-like related-protein activity and/or functionat all.

(48) A eukaryotic cell comprising

-   -   a modified ssn6-like gene or a modified ssn6-like related gene,        wherein said gene has inserted a foreign nucleotide sequence, to        the effect that    -   (i) the eukaryotic cell is not able to provide an SSN6-like        protein or an SSN6-like related protein that exerts its wildtype        function and/or wildtype activity, preferably with respect to        its activity and/or function in regulating the LLP promoter,    -   (ii) the amount of SSN6-like protein or of SSN6-like related        protein being present in the eukaryotic cell differs from the        amount of SSN6-like protein or of SSN6-like related protein        being present in its wildtype form, preferably the amount of the        SSN6-like protein or of the SSN6-like related protein is        reduced, and/or    -   (iii) no SSN6-like protein or SSN6-like related protein is        present in the eukaryotic cell,    -   a gene of interest under control of the LLP promoter, and    -   an llp gene,

preferably wherein said eukaryotic cell exhibits reduced SSN6-likeprotein or SSN6-like related-protein activity and/or function, or noSSN6-like protein or SSN6-like related-protein activity and/or functionat all.

(49) Nucleotide sequence comprising at it's 5′-end a nucleotide sequencewhich codes for the peptide sequence of the LLP-signal sequence andwhich is depicted in SEQ ID NO: 3, and further comprising a nucleotidesequence which is coding for a protein different to the nativeLLP-protein sequence.

(50) Use of a nucleotide sequence according to item (45) for themanufacture of a non-LLP-protein in a yeast cell, which yeast cellsecretes said non-LLP-protein into the cell culture medium, due to thesecretion-promoting activity of said peptide sequence of the LLP-signalsequence which is depicted in SEQ ID NO: 3.

(51) Use of a promotor comprising the LLP-promoter sequence according toSEQ ID NO: 12 (1000 bp of LLP-promoter), or according to SEQ ID NO: 2(605 bp of LLP-promoter), or according to SEQ ID NO: 129 (576 bp ofLLP-promoter), or according to SEQ ID NO: 130 (512 bp of LLP-promoter),or according to SEQ ID NO: 131 (472 bp of LLP-promoter), or according toSEQ ID NO: 132 (404 bp of LLP-promoter), or according to SEQ ID NO: 133(372 bp of LLP-promoter), or according to SEQ ID NO: 134 (305 bp ofLLP-promoter), in a vector for expression of a gene of interest, whereinsaid LLP-promoter sequence has at least 30%, preferably 40%, preferably50%, preferably 60%, preferably 70%, preferably, preferably 80%,preferably 90%, preferably 95%, preferably 97%, preferably 99%,preferably 100% sequence identity with SEQ ID NO: 12.

(52) Vector comprising the LLP-promoter sequence as defined in item(51).

(53) Host cell comprising a vector according to item (52) or comprisingone or more of the LLP-promoter sequences according to item (51).

(54) Nucleic acid comprising one or more of the LLP-promoter sequencesaccording to item (51). In a preferred embodiment, the nucleic acidaccording to item (54) is used as a promoter.

Definitions of Terms as Used within the Meaning of the Present Invention

Within the meaning of the present invention, the SSN6-like protein is aprotein that comprises, preferably consists of, the amino acid sequenceas depicted in SEQ ID NO: 8.

The term “ssn6-like gene” denotes a gene encoding the SSN6-like protein.

The coding region of the ssn6-like gene of P. pastoris is depicted inSEQ ID NO: 1.

Genes or proteins that resemble the ssn6-like gene or SSN6-like proteinin respect of function, activity and sequence, or only in respect offunction and activity, are denoted herein as “ssn6-like related genes”or “ssn6-like related proteins”.

Within the meaning of the present invention, the term “gene” denotes acertain nucleic acid sequence that encodes a polypeptide or an RNA chainthat has a function in the organism. The nucleic acid sequence comprisesregulatory regions (herein also denoted as regulatory polynucleotidesequences or regulatory sequences), transcribed regions (such as regionsthat code for proteins, but also regions that are transcribed but do notcode for proteins, such as introns) and/or other functional sequenceregions.

In general, regulatory regions are primarily regions flanking the 3′-and 5-region of the coding region of the gene, but certain regulatoryregions such as Trans-regulatory elements, might be located quitedistant within the same chromosome or might even be located on adifferent chromosome. In particular, within the meaning of the presentinvention, the regulatory regions of the ssn6-like (or ssn6-likerelated) gene comprise the flanking region being located up to 200nucleotides up- and downstream of the 3′- and 5′-end of the ssn6-likecoding region. Examples of regulatory polynucleotide sequences arepromoters, enhancers, terminators, silencers, IRES-sequences,ribosome-binding sites, and sequences stabilizing or destabilizing themRNA by secondary structures. Within the meaning of the presentinvention, the term “regulatory polynucleotide sequence” denotespolynucleotide sequences that modify the expression of genes, forexample the ssn6-like gene and/or of the ssn6 gene, and/or a gene ofinterest and/or the llp gene.

“Protein-antigens that are suitbale for vaccines” are antigens that areable to elicit a proper, desired immune response upon vaccination. Forinstance, such antigens are neuraminidase (NA) or haemagglutinin (HA) ofinfluenza virus. Further antigens that are suitbale for vaccinationpurpose are known to a person skilled in the art. Methods for testingthe suitability of proteins for eliciting a proper immune response areknown to a skilled person.

A “polynucleotide” or “nucleic acid” sequence includes DNA(desoxyribonucleic acid) or RNA (ribonucleic acid), in single strandedor double stranded form or otherwise.

In general, the term “ssn6-like gene” denotes a gene encoding theSSN6-like protein. The ssn6-like gene belongs to an evolutionaryconserved family of proteins (see FIG. 9 A), and it is known thatcertain yeasts, flies, worms and mammals contain proteins that resembleSSN6-like proteins in sequence and function/activity. These proteins aredenoted herein as “SSN6-like related proteins”, and the genes thatencode these proteins are denoted herein as “ssn6-like related genes”.Examples of yeasts that contain proteins that belong to the SSN6-likerelated protein family are Pichia pastoris (P. Pastoris), Saccharomycescerevisiae (S. cerevisiae) and Candida albicans (C. albicans).

Within the meaning of the present invention, the term “ssn6-like-relatedprotein” for example denotes the respective proteins whose NCBI(National Center for Biotechnology Information, USA) GenBank accessionnumbers are noted on the left hand site of FIG. 9A. FIG. 9A only showsan internal part of these sequences aligned to the correspondingsequence part of the P. Pastoris ssn6-like sequence (NCBI GenBankaccession number CCA36593.1). In detail these proteins includeXP_004181958.1 (SEQ ID NO: 24), KDQ17717.1 (SEQ ID NO: 25), CCK71477.1(SEQ ID NO: 26), EDK38165.2 (SEQ ID NO: 27), XP_003688172.1 (SEQ ID NO:28), XP_003667908.1 (SEQ ID NO: 29), EGA59684.1 (SEQ ID NO: 30),EDN64727.1 (SEQ ID NO: 31), AAA34545.1 (SEQ ID NO: 32), NP_009670.3 (SEQID NO: 33), XP_001731010.1 (SEQ ID NO: 34), CCU98386.1 (SEQ ID NO: 35),XP_646078.1 (SEQ ID NO: 36), XP_003288629.1 (SEQ ID NO: 37), CCF50299.1(SEQ ID NO: 38), XP_761648.1 (SEQ ID NO: 39), XP_007880878.1 (SEQ ID NO:40), EPB82504.1 (SEQ ID NO: 41), CDK26448.1 (SEQ ID NO: 42), ESW97404.1(SEQ ID NO: 43), CCH42354.1 (SEQ ID NO: 44), CDR41214.1 (SEQ ID NO: 45),XP_002489776.1 (SEQ ID NO: 46), XP_002770760.1 (SEQ ID NO: 47),EDK37317.2 (SEQ ID NO: 48), XP_001485744.1 (SEQ ID NO: 49),XP_002619527.1 (SEQ ID NO: 50), XP_004200097.1 (SEQ ID NO: 51),XP_004199242.1 (SEQ ID NO: 52), XP_002419644.1 (SEQ ID NO: 53),BAF31137.1 (SEQ ID NO: 54), XP_719833.1 (SEQ ID NO: 55), EMG49052.1 (SEQID NO: 56), XP_002551300.1 (SEQ ID NO: 57), XP_001526425.1 (SEQ ID NO:58), CCE42279.1 (SEQ ID NO: 59), XP_003868368.1 (SEQ ID NO: 60),XP_001387682.2 (SEQ ID NO: 61), XP_007376632.1 (SEQ ID NO: 62) as foundin the NCBI data base under http://www.ncbi.nlm.nih.gov/nucleotide/.

Within the meaning of the present invention, the term “SSN6-likeprotein” denotes the protein comprising (preferably consisting of) theamino acid sequence as depicted in the sequence depicted in FIG. 2A (SEQID NO: 8).

Furthermore within the meaning of the present invention, the term“SSN6-like protein” denotes a protein comprising consensus amino acidsequences depicted in FIG. 9 B or 9 C, namely:

FIG. 9 B: Consensus sequence 1 (SEQ ID NO: 63)W(CGL)(SLTA)(IMV)G(VINSTK)LY(YFLA)(QNSRKE)(INLM)(GNSKR)Q(NFYL)(HRETPAK)D(AST)(LI) (DGTNASE)(AV)(YF) and/orFIG. 9 C: Consensus sequence 2 (SEQ ID NO: 64)W(YFLED)(NDG)L(GLAS)(TSIQC)(LIV)YE(TSARKQ)(CS)(NHSDEH)(DNK-RGF)(Q-H)(ILTHVAS)(TSNQERIAMG)D(ASV)(LIASC)(DHNE)(SA)(YC) (EAKRQMTLNDS)(RQK)

The both consensus sequences are written in single letter code andgroups of amino acids in brackets. Groups of amino acids in bracketsmeans that either amino acid in the brackets can be present at thatposition within the consensus sequence. For example the consensussequence in FIG. 9 B starts with W at position 1, and at position 2there can be present either C or G or L, at position 3 there can bepresent either S or L or T or A, etc.

The corresponding amino acid position within the context of the P.pastoris SSN6-like protein is given below the consensus sequences ofFIGS. 9 B and 9 C, e.g. the Tryptophane residue (W) in position 1 of theconsensus sequence in FIG. 9 B corresponds to the amino acid position352 of the P. pastoris SSN6-like protein sequence.

In case there is written a “-” (dash), for example at position 407 ofconsensus sequence in FIG. 9 C, (Q-H), this means that on this positionthere can be either a Q, no amino acid, or a H.

Further within the meaning of the present invention, the term “ssn6-likerelated gene” or “SSN6-like related protein” denotes a gene or proteinthat resembles the ssn6-like gene or SSN6-like protein in respect offunction, activity and sequence, or only in respect of function andactivity.

The coding region of the ssn6-like gene of P. Pastoris is depicted inFIG. 1A (SEQ ID NO: 1).

The amino acid sequence of the SSN6-like protein of P. Pastoris isdepicted in FIG. 1 J (SEQ ID NO: 13).

The term “llp gene” denotes a gene encoding LLP protein. SEQ ID NO: 16depicts the nucleotide sequence of the coding region of the llp genefrom P. Pastoris, including the signal sequence.

“Resembling with respect to function” or “resembling with respect toactivity” defines that the ssn6-like related gene product, i.e. theSSN6-like related protein that is encoded by the “ssn6-like relatedgene”, exhibits essentially the same function and activity as theSSN6-like protein of P. Pastoris (depicted in SEQ ID NO: 13) with regardto the LLP-promoter (“Lectin-like protein with similarity to Flo1p”),when said function and activity is compared in a suitable system whenapplying comparable, preferably corresponding, conditions. The functionand activity that is of interest in the present invention, and thus hasto be compared or assessed, respectively, is the ability of theSSN6-like protein and SSN6-like related protein, respectively, toregulate, preferably repress (reduce), and even more preferably prevent,the expression of genes that are under control of the LLP-promoter. Forinstance, in a wildtype situation, typically the gene that is undercontrol of the LLP promoter is a gene encoding LLP protein. In thiscase, in order to determine whether the SSN6-like related proteinexhibits essentially the same function and/or activity as the SSN6-likeprotein, for instance the amount of LLP protein being present iscompared, i.e. the amount of LLP protein in an expression system whereinan ssn6-like related gene (and, thus, an SSN6-like related protein) ispresent, compared to an expression system wherein an SSN6-like gene(and, thus, an SSN6-like protein) is present. If it is shown that thecandidate ssn6-like related gene and the ssn6-like gene (and its geneproducts, (candidate) SSN6-like related protein and SSN6-like protein,respectively (also referred to herein as “SSN6-like/SSN6-like relatedprotein” or “SSN6-like related/SSN6-like protein”) exhibit essentiallythe same function/activity with regard to the LLP-promoter (and withregard to the gene that is under control of said promoter, such as thellp gene), e.g. with regard to the determined amount of LLP protein,then the candidate ssn6-like related gene is a ssn6-like related genewithin the meaning of the present invention.

A promoter is defined as a DNA regulatory region capable of binding anRNA polymerase in a cell and that initiates transcription of aparticular gene to which it operably links. Typically, promoters arelocated near the transcription start sites of the respective gene(s)that is under control of said promoter, on the same strand. FIG. 1 B(SEQ ID NO: 2) and FIG. 1 I (SEQ ID NO: 12) show nucleotide sequences ofthe LLP promoter of P. Pastoris (FIG. 1 B=first 605 nucleotides 5′ fromATG start codon, FIG. 1 I=fist 1000 nucleotides 5′ from ATG startcodon).

Whenever reference is made herein to an LLP promoter, an “LLP-likepromoter” is also comprised by this expression. Within the meaning ofthe present invention, an LLP-like promoter is a promoter that exhibitsessentially the same function/activity as the LLP promoter, i.e. isstill able to essentially exert the LLP promoter's wildtypefunction/activity.

In order to determine whether a candidate ssn6-like-related gene andSSN6-like-related protein, respectively, exhibits essentially the samefunction and activity as the ssn6-like-gene and SSN6-like protein,respectively, with respect to the LLP-promoter, any suitable method thatis known to a person skilled in the art can be used. Examples of suchmethods are measuring the amount of LLP protein in the supernatant of acell culture for example by Enzyme Linked Inmmunosorbent Assay (ELISA),or by western blotting, or measuring the level of LLP mRNA by northernblotting or by quantitative Polymerase Chain Reaction (qPCR) or reversetranscriptase qPCR, or measuring the activity of the LLP-promoter orLLP-like promoter (“LLP/LLP-like promoter”) for example by usingluciferase reporter gene assays or by using LLP-promoter-greenfluorescent protein (GFP) constructs, etc. All these methods are wellknown to a person skilled in the art and represent routine work. Atextbook comprising protocols for routine methods is for instanceSambrook et al., “Molecular Cloning: A Laboratory Manual”, 4^(th)Edition, Cold Spring Harbor Laboratory Press, (2012), referred to hereinas Sambrook et al. Within the meaning of the present invention, acandidate ssn6-like-related gene is considered as exhibiting essentiallythe same function/activity as the ssn6-like-gene, if its effect (and theeffect of its gene product, respectively) on the LLP protein expressionis essentially the same.

Within the meaning of the present invention, the expression “essentiallythe same” defines a deviation of up to 20%, preferably of up to 10%,more preferably of up to 7% and even more preferably of up to 3% of agiven value.

Accordingly, the expression “essentially no” defines that there is anamount/activity of the respective matter (such as protein) present (orleft) that corresponds to at most 20%, preferably at most 10%, morepreferably at most 7%, and even more preferably at most 3% of therespective wildtype amount/activity. “Essentially no” also includes theabsence of the respective matter/amount/activity, preferably meaningbelow the detection limit of the methods described in this applicationfor that matter/amount/activity.

Within the meaning of the present invention the term “reduced”, e.g.“reduced” activity, “reduced” function, or “reduced” amount denotes thatthe total activity, function, preferably gene regulatory function, oramount of a matter (e.g. SSN6-like protein or SSN6-like related proteinof the modified eukaryotic cell; or LLP promoter) is at most 20%,preferably at most 15%, more preferably at most 10%, even morepreferably at most 5% of the total activity, function and/or amount ofthe wildtype form of this matter (e.g. SSN6-like protein or SSN6-likerelated protein; or LLP promoter).

It is possible that nucleotide sequences and the proteins they encode,respectively, which exhibit essentially the same function and activityas ssn6-like gene and SSN6-like protein, respectively, exhibit acomparably low degree of sequence identity such as 50%, 40% or 30% oreven lower, such as 25%, 20%, 19%, 18%, 17%, 16%, 15%, 12%, 10%, or 5%.Thus, ssn6-like-related genes/SSN6-like-related proteins within themeaning of the present invention are genes/proteins that—first ofall—exhibit essentially the same function/activity as the ssn6-likegene/SSN6-like protein with regard to the LLP protein (in wild typesituation). Hence, in a preferred embodiment of the present invention,the term “ssn6-like-related gene” denotes genes that resemble thessn6-like gene as defined herein in respect of function/activity, i.e.the ability of the SSN6-like-related protein being encoded by thessn6-like-related gene to regulate the expression of genes that areunder control of the LLP-promoter, and LLP-promoter sequence,respectively.

It is also possible that an SSN6-like related protein comprises theconsensus amino acid sequences depicted in FIG. 9 B and/or FIG. 9C.

In a further preferred embodiment of the present invention, the“ssn6-like related gene” resembles the ssn6-like gene as defined hereinnot only in respect of function/activity, i.e. the ability of theSSN6-like related protein being encoded by the ssn6-like related gene toregulate the expression of genes that are under control of theLLP-promoter or modified LLP promoter, and LLP-promoter sequence,respectively, but additionally originates from a microorganism beingselected from the group consisting of Komagataella pastoris CBS 7435(Synonym/other names: Pichia pastoris, Pichia pastoris CBS 7435),Komagataella pastoris GS115 (Synonym/other names: Pichia pastoris,Pichia pastoris GS115), Scheffersomyces stipitis CBS 6054 (Synonym/othernames: Pichia stipitis, Pichia stipitis CBS 6054), Millerozyma farinosaCBS 7064 (other name: Pichia farinosa CBS 7064), Candida parapsilosis,Candida orthopsilosis Co 90-125, Debaryomyces hansenii CBS767,Spathaspora passalidarum NRRL Y-27907, Candida albicans, Candidaalbicans, Candida albicans SC5314, Candida maltosa Xu316, Candidatropicalis MYA-3404 (other name: Candida tropicalis T1), Lodderomyceselongisporus NRRL YB-4239 (other name: Saccharomyces elongisporus),Clavispora lusitaniae ATCC 4272 (genebank anamorph: Candida lusitaniaeATCC 42720), Meyerozyma guilliermondii ATCC 6260 (genebank anamorph:Pichia guilliermondii ATCC 6260), Wickerhamomyces ciferrii, Ogataeaparapolymorpha DL-1 (synonym and other names: Hansenula polymorpha,Hansenula polymorpha DL-1, Ogataea angusta DL-1, Ogataea parapolymorphaATCC 26012, Ogataea parapolymorpha DL-1, Pichia angusta DL-1),Cyberlindnera fabianii (synonyms and other names: Hansenula fabianii,Pichia fabianii, . . . ) Kuraishia capsulata CBS 1993, Dictyosteliumdiscoideum AX4 (belongs to social amoebae), Tetrapisispora phaffii CBS4417 (synonym: Fabospora phaffii, Dictyostelium purpureum (belongs tosocial amoebae), Pseudozyma flocculosa PF-1, Malassezia globosa CBS7966, Botryobasidium botryosum FD-172 SS1 (basidiomycete), Naumovozymadairenensis CBS 421 (synonyme: Saccharomyces dairenensis),Tetrapisispora blattae CBS 6284, Mucor circinelloides f. circinelloides1006PhL (Early diverging fungal lineage), Malassezia sympodialis ATCC42132, Kazachstania naganishii CBS 8797 (Saccharomyces naganishii),Saccharomyces cerevisiae YJM789, Saccharomyces cerevisiae FostersB,Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Ustilagohordei (Corn smut fungus, basidiomycete), Meyerozyma guilliermondii ATCC6260 (synonym/other names: Candida guilliermondii, Pichia guilliermondiiATCC 6260), and Ustilago maydis 521 (Corn smut fungus, basidiomycete).

In a preferred embodiment, the “modified ssn6-like gene” and “modifiedssn6-like related gene”, and the respective proteins these genes encode,do not correspond to/are not derived from Saccharomyces cerevisiae ssn6or Tup1 nucleotide or amino acid sequences, especially do not correspondto the following sequences:

SEQ ID NO: 135 (nucleotide sequence encoding SSN6)

SEQ ID NO: 136 (nucleotide sequence encoding Tup1)

SEQ ID NO: 137 (amino acid sequence of SSN6)

SEQ ID NO: 138, (amino acid sequence of Tup1).

In a further preferred embodiment, the modified eukaryotic cellcomprises modified ssn6-like genes or ssn6-like related genes, but noother modified gene(s) that regulate(s) the expression of a gene that isunder control of a promoter that is repressed in the presence ofSSN6-like related protein or SSN6-like protein.

“Sequence identity” or “% identity” refers to the percentage of residuematches between at least two polypeptide or polynucleotide sequencesaligned using a standardized algorithm. Such an algorithm may insert, ina standardized and reproducible way, gaps in the sequences beingcompared in order to optimize alignment between two sequences, andtherefore achieve a more meaningful comparison of the two sequences. Forpurposes of the present invention, the sequence identity between twoamino acid sequences or nucleotide is determined using the NCBI BLASTprogram version 2.2.29 (Jan. 6, 2014) (Altschul et al., Nucleic AcidsRes. (1997) 25:3389-3402). Sequence identity of two amino acid sequencescan be determined with blastp set at the following parameters: Matrix:BLOSUM62, Word Size: 3; Expect value: 10; Gap cost: Existence=11,Extension=1; Filter=low complexity activated; Filter String: L;Compositional adjustments: Conditional compositional score matrixadjustment. For purposes of the present invention, the sequence identitybetween two nucleotide sequences is determined using the NCBI BLASTprogram version 2.2.29 (Jan. 6, 2014) with blastn set at the followingexemplary parameters: Word Size: 11; Expect value: 10; Gap costs:Existence=5, Extension=2; Filter=low complexity activated;Match/Mismatch Scores: 2,−3; Filter String: L; m.

Nucleic acid sequences alternatively can be characterized by theirdegree of complementarity. As used herein, the term “complementary”refers to the ability of purine and pyrimidine nucleotide sequences toassociate through hydrogen bonding to form double-stranded nucleic acidmolecules. Guanine and cytosine, adenine and thymine, and adenine anduracil are complementary and can associate through hydrogen bondingresulting in the formation of doublestranded nucleic acid molecules whentwo nucleic acid molecules have “complementary” sequences. Thecomplementary DNA sequences are referred to as a “complement.” Inaccordance with the invention “highly stringent conditions” meanshybridization at 65° C. in 5×SSPE and 50% formamide, and washing at 65°C. in 0.5×SSPE. Conditions for high stringency hybridization aredescribed in Sambrook et al., “Molecular Cloning: A Laboratory Manual”,3rd Edition, Cold Spring Harbor Laboratory Press, (2001), incorporatedherein by reference. In some illustrative aspects, hybridization canoccur along the full-length of the isolated nucleic acid, or along partof its length, or to a fragment thereof.

The following list indicates examples of organisms that comprisessn6-like related genes: Komagataella pastoris CBS 7435 (Synonym/othernames: Pichia pastoris, Pichia pastoris CBS 7435), Komagataella pastorisGS115 (Synonym/other names: Pichia pastoris, Pichia pastoris GS115),Scheffersomyces stipitis CBS 6054 (Synonym/other names: Pichia stipitis,Pichia stipitis CBS 6054), Millerozyma farinosa CBS 7064 (other name:Pichia farinosa CBS 7064), Candida parapsilosis, Candida orthopsilosisCo 90-125, Debaryomyces hansenii CBS767, Spathaspora passalidarum NRRLY-27907, Candida albicans, Candida albicans, Candida albicans SC5314,Candida maltosa Xu316, Candida tropicalis MYA-3404 (other name: Candidatropicalis T1), Lodderomyces elongisporus NRRL YB-4239 (other name:Saccharomyces elongisporus), Clavispora lusitaniae ATCC 4272 (genebankanamorph: Candida lusitaniae ATCC 42720), Meyerozyma guilliermondii ATCC6260 (genebank anamorph: Pichia guilliermondii ATCC 6260),Wickerhamomyces ciferrii, Ogataea parapolymorpha DL-1 (synonym and othernames: Hansenula polymorpha, Hansenula polymorpha DL-1, Ogataea angustaDL-1, Ogataea parapolymorpha ATCC 26012, Ogataea parapolymorpha DL-1,Pichia angusta DL-1), Cyberlindnera fabianii (synonyms and other names:Hansenula fabianii, Pichia fabianii, . . . ), Kuraishia capsulata CBS1993, Dictyostelium discoideum AX4 (belongs to social amoebae),Tetrapisispora phaffii CBS 4417 (synonym: Fabospora phaffii,Dictyostelium purpureum (belongs to social amoebae), Pseudozymaflocculosa PF-1, Malassezia globosa CBS 7966, Botryobasidium botryosumFD-172 SS1 (asdmVcete), Naumovozyma dairenensis CBS 421 (synonyme:Saccharomyces dairenensis), Tetrapisispora blattae CBS 6284, Mucorcircinelloides f. circinelloides 1006PhL (Early diverging fungallineage), Malassezia sympodialis ATCC 42132, Kazachstania naganishii CBS8797 (Saccharomyces naganishii), Saccharomyces cerevisiae YJM789,Saccharomyces cerevisiae FostersB, Saccharomyces cerevisiae,Saccharomyces cerevisiae S288c, Ustilago hordei (Corn smut fungus,basidiomycete), Meyerozyma guilliermondii ATCC 6260 (synonym/othernames: Candida guilliermondii, Pichia guilliermondii ATCC 6260),Ustilago maydis 521, (Corn smut fungus, basidiomycete).

Within the meaning of the present invention, the term “wildtype” denotesthat a certain matter is present in its natural occurring state, andthus exerts its natural function and/or activity. A “certain matter” canfor instance be an organism such as a microorganism, a cell, or,preferably, a certain protein such as the SSN6 protein or the SSN6-likeprotein. The wildtype state, or wildtype form, respectively, of acertain matter is distinguishable from mutant forms of said matter, asfor instance structural mutant forms can result from modifications thatare carried out artificially, such as by in vitro, in vivo or ex vivomodifications or manipulations. Modifications that can be carried outartificially are described elsewhere herein. An organism that comprisesmodifications is “modified” compared to its wildtype form with regard tothe modified structure.

Within the meaning of the present invention, the term “function” definesthe physiological role of a certain matter, such as the SSN6-likeprotein or SSN6-like related protein, and the term “activity” defines towhich extent said certain matter exerts its function. An example of aphysiological role of a certain matter is the regulating, preferablyrepressing, role of SSN6-like protein or SSN6-like related protein, onthe activity of the LLP-promoter, and hence, on the expression of genesthat are under control of said promoter.

Within the meaning of the present invention, the term “wildtypefunction” denotes the function a certain structure or matter (such as anorganism, a protein, or a gene) exerts in its natural state.Accordingly, the term “wildtype activity” denotes the activity a certainstructure exerts in its natural state.

Within the meaning of the present invention, an organism can be modifiedonly with regard to a single specific structural and/or functionalfeature, or with regard to multiple structural and/or functionalfeatures. Specific structural and/or functional features that aremodified are features that modulate (i.e. influence, either positivelyor negatively, preferably negatively) the expression of the ssn6 gene orssn6-like gene.

An example of a specific feature that can be modified is for instancethe nucleotide sequence of ssn6-like gene or ssn6-like related gene,with this gene comprising a coding sequence and a regulatory sequencethat influences the expression of said genes as disclosed elsewhereherein.

Regulatory sequences are sequences that control the expression ofcertain genes. Within the meaning of the present invention, saidregulatory sequences are for example sequences that control theexpression of the ssn6-like gene, the ssn6-like related gene or theLLP-gene, such as promoters, enhancers, terminators, silencers,IRES-sequences, ribosome-binding sites, and sequences stabilizing ordestabilizing the mRNA by secondary structures.

Within the meaning of the present invention, the term “heterologousnucleotide sequence” is a polynucleotide that does not naturally occurin the cell, e.g. because the nucleotide sequence of the polynucleotidedoes not naturally occur in eukaryotic cells, such as eukaryotic cellsas defined elsewhere herein.

Within the meaning of the present invention, the names “Komagataellapastoris” and “Pichia pastoris” are synonymous.

The names “NRRL Y-11430, CBS7435” are synonymously used in the presentinvention. The genome of CBS7435 comprises chromosomes 1 to 4 and amitochondrium. The respective nucleotide sequences have the followingGenBank Accession Numbers: Chromosome 1: FR839628.1; Chromosome 2:FR839629.1; Chromosome 3: FR839630.1; Chromosome 4: FR839631.1;Mitochondrium: FR839632.1 (Publication: High-quality genome sequence ofPichia pastoris CBS7435, Kuberl A, Schneider J, Thallinger G G, AnderlI, Wibberg D, Hajek T, Jaenicke S, Brinkrolf K, Goesmann A,Szczepanowski R, Puhler A, Schwab H, Glieder A, Pichler H, J Biotechnol,volume 154 issue 4, pages 312-320 year 2011).

A “vector” is a replicon, such as plasmid, phage, bacterial artificialchromosome (BAC) or cosmid, into which another DNA segment (e.g. aforeign gene) may be inserted so as to bring about the replication ofsaid inserted DNA segment, resulting in expression of said insertedsequence. Vectors may comprise a promoter and one or more controlelements (e.g., enhancer elements) that are homologous or heterologousto said inserted DNA segment but are recognized and used by the hostcell. A “replicon” is any genetic element (e.g., plasmid, chromosome,virus) that functions as a unit of DNA replication within a cell. Areplicon can be an autonomous unit. This means that it is capable ofreplication under its own control. Within the meaning of the presentinvention, the vector comprises a promoter, wherein said promoter ischaracterized in that it is repressed in the presence of SSN6-likerelated protein or SSN6-like protein if said vector is introduced into asuitable host cell. In a preferred embodiment of the present invention,the vector comprises a promoter for the coding sequence of theLLP-protein and/or for the coding sequence of a protein of interest,wherein said promoter is characterized in that it is repressed in thepresence of SSN6-like related protein or SSN6-like protein if saidvector is introduced into a suitable host cell.

A common type of vector is a “plasmid”, which generally is aself-contained molecule of double-stranded DNA, usually of bacterialorigin, that can readily accept additional (foreign) DNA and which canbe readily introduced into a suitable host cell. In general, vectorsenable the introduction of nucleotide sequences into a host cell, so asto transform the host and, optionally, to promote expression and/orreplication of the introduced sequence.

A vector often contains coding DNA and regulatory sequences such aspromoter DNA and has one or more restriction sites suitable forinserting additional, e.g. foreign such as heterologous, DNA. PromoterDNA and coding DNA may be from the same gene or from different genes,and may be from the same or different organisms. Vectors, such asrecombinant cloning vectors, will often include one or more replicationsystems for cloning or expression, one or more markers for selection inthe host, e.g. antibiotic resistance, and one or more expressioncassettes. Vector, or vector constructs, respectively, may be producedusing conventional molecular biology and recombinant DNA techniqueswithin the skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, 4^(th) Edition (2012) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 2012”); DNACloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985);F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology,John Wiley & Sons, Inc. (1994).

Generally, the specific structural elements of a vector depend on theintended use of said vector. For instance, in order to propagate avector in a host cell, it may contain one or more “origins ofreplication” sites (often also termed “ori”), which is a specificnucleic acid sequence at which replication is initiated. Accordingly,the term “origin of replication” or “ori” refers to a nucleic acidsequence that initiates nucleic acid replication.

“Ori T” refers to an “origin of transfer” that permits the transfer ofthe vector from one bacterial cell to another.

Dependent on in which organism/cell the (expression) vector is intendedfor being used, a person skilled in the art knows which markers have tobe present in the vector. If, for instance, the vector is intended forbeing used in yeast, amongst others, commonly used yeast markers arepresent, such as beta-galactosidase, Zeocin, Geneticin, URA3, HIS3,LEU2, TRP1 and LYS2. If the vector is intended for being used inbacteria such as E. coli, amongst others an on (see above) and/orselectable markers such as genes conferring antibiotic resistance can bepresent.

Suitable selectable markers depend on the respective system that is usedand are known to a person skilled in the art. Transformedmicroorganisms, that is, those containing recombinant molecules such asa vector (expression vector) or plasmid, may be selected with a varietyof positive and/or negative selection methods or markers. For instance,a positive selection marker can be a gene that allows growth in theabsence of an essential nutrient, such as an amino acid. A variety ofsuitable positive/negative selection pairs are available and known inthe art. For example, various amino acid analogs known in the art couldbe used as a negative selection, while growth on minimal media (relativeto the amino acid analog) could be used as a positive selection.Visually detectable markers are also suitable for being uses in thepresent invention, and may be positively and negatively selected and/orscreened using technologies such as fluorescence activated cell sorting(FACS) or microfluidics. Examples of detectable markers include variousenzymes, prosthetic groups, fluorescent markers, luminescent markers,bioluminescent markers, and the like. Examples of suitable fluorescentproteins include, but are not limited to, yellow fluorescent protein(YFP), green fluorescence protein (GFP), cyan fluorescence protein(CFP), umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichiorotriazinylamine fluorescein, dansyl chloride,phycoerythrin and the like. Examples of suitable bioluminescent markersinclude, but are not limited to, luciferase (e.g., bacterial, firefly,click beetle and the like), luciferin, aequorin and the like. Examplesof suitable enzyme systems having visually detectable signals include,but are not limited to, galactosidases, glucorinidases, phosphatases,peroxidases, cholinesterases and the like. In other aspects, thepositive selection marker is a gene that confers resistance to acompound, which would be lethal to the cell in the absence of the gene.For example, a cell expressing an antibiotic resistance gene wouldsurvive in the presence of an antibiotic, while a cell lacking the genewould not. For instance, the presence of a tetracycline resistance genecould be positively selected for in the presence of tetracycline, andnegatively selected against in the presence of fusaric acid. Suitableantibiotic resistance genes include, but are not limited to, genes suchas ampicillin-resistance gene, neomycin-resistance gene,blasticidin-resistance gene, hygromycin-resistance gene,puromycin-resistance gene, chloramphenicol-resistance gene,apramycin-resistance gene, geneticin-resistance gene, zeocin-resistancegene, and the like. In certain aspects, the negative selection marker isa gene that is lethal to the target cell in the presence of a particularsubstrate. For example, the thyA gene is lethal in the presence oftrimethoprim. Accordingly, cells that grow in the presence trimethoprimdo not express the thyA gene.

In order to purify the recombinant proteins, the respective (expression)vectors used often comprise suitable purification sequences(purification markers). For instance, a vector may comprise a C-terminalc-myc epitope and polyhistidine sequence for detection and purificationof the recombinant protein(s).

As described elsewhere herein, a vector can be used for introducing anucleic acid sequence into a host cell, or into the host cell's genome,such as the genome of a yeast such as P. pastoris, or of a bacterium,such as E. coli. The introduction of (heterologous) nucleic acid into ahost cell's nucleic acid is denoted as “recombination”. Such arecombination process can be targeted, i.e. take place at a defined,desired site of the genome (nucleic acid sequence) it is to beintroduced. In this case, the recombination is denoted as “homologousrecombination” (or “targeted recombination”/“targeted integration”).Also, the recombination process can be random (“randomrecombination”/“random integration”). In this case, the recombination isnon-homologous.

The term “homologous recombination” refers to a type of geneticrecombination, a process of physical rearrangement occurring between twodifferent strands of DNA molecules. Homologous recombination involvesthe alignment of identical or similar sequences, a crossover between thealigned homologous DNA strands of the two molecules, and breaking andrepair of the DNA to produce an exchange of material between thestrands. Homologous recombination is distinguished from other types ofrecombination. For example, “site specific recombination”, asexemplified by invertible elements, resolvases, and some phageintegration events are examples of non-homologous recombination. Thoughin many cases identical or similar sequences are required at the tworecombining sites, the sequences are short, distinguishing them from thelonger stretches (hundreds of base pairs) used in homologousrecombination. (J Rubnitz and S Subramani. 1984, Mol Cell Biol. 4:2253-2258).

If the vector aligns in a non-homologous region of the target nucleicacid, the recombination is random.

Within the meaning of the present invention, in connection withhomologous recombination, two DNA sequences are “substantiallyhomologous” if they are able to mediate a homologous recombination. Thismay for instance the case when at least about 80%, preferably at leastabout 90% or 95%, more preferably at least about 97% or 98% of thenucleotides match over a defined length of the DNA sequences, asdetermined by sequence comparison algorithms known to a person skilledin the art and described elsewhere in this application. It is alsopossible that two DNA sequences exhibit 100% match of the nucleotidesover a defined length. In this case, the two DNA sequences arehomologous to each other. Within the meaning of the present invention, aDNA sequence in the vector, e.g. the expression vector, is substantiallyhomologous, or homologous, to a desired target integration site in thehost cell.

Within the meaning of the present invention, bothintegration/recombination events can occur, i.e. the homologousrecombination and the random recombination. For instance, if it isdesired to replace a certain gene (or coding sequence) by the gene ofinterest, then a homologous recombination will be carried out. Withinthe meaning of the present invention, it may be desired to replace agene that is under the control of a promoter that is repressable bySSN6-like related or SSN6-like protein, such as a nucleic acid sequenceencoding LLP protein (“Option 1” in FIG. 3 C).

Within the meaning of the present invention it is however also possiblethat it may be desired to not replace a gene that is under the controlof a promoter that is repressable by SSN6-like related or SSN6-likeprotein, such as a nucleic acid sequence encoding LLP protein, but toexpress said gene (such as the gene encoding LLP), and to additionallyexpress the gene of interest (“Option 2” in FIG. 3 D). In such a case, arandom (heterologous) recombination is carried out.

Within the meaning of the present invention, the term “gene of interest”(GOI) refers to a gene that is intended for being expressed in a hostcell. In the present invention, genes of interest are genes selected forexample from the group encoding enzymes, antibodies or fragmentsthereof, hormones, structural proteins (e.g. albumin) andprotein-antigens present in vaccines.

Within the meaning of the present invention, the term “expressioncassette” refers to a part of vector DNA that is for instance used forcloning and/or transformation. An expression cassette comprises one ormore genes and sequences controlling their expression. An expressioncassette can be inserted in a nucleotide sequence such as a genome forinstance by means of homologous recombination or by heterologousrecombination.

Within the meaning of the present invention, the term “host cell”denotes any cell that, under suitable conditions, is capable ofpropagating or expressing a vector such as an expression vector. Forinstance, in the present invention an expression vector can comprise apromoter, wherein said promoter is characterized in that it is repressedin the presence of SSN6-like protein or SSN6-like related protein.

The host cell that comprises a vector can be any suitable host cell,such as a bacterial cell, preferably an E. coli cell. It is alsopossible that the vector, such as the expression vector, is present in aeukaryotic cell. In this case, the host cell is said eukaryotic cell.If, as described elsewhere herein, said eukaryotic cell is modified withregard to the ssn6-like gene, ssn6-like related gene, SSN6-like proteinor SSN6-like related protein, the eukaryotic cell is denoted as“modified eukaryotic cell”. In a preferred embodiment of the presentinvention, the eukaryotic cell is a fungal cell, preferably a yeastcell, more preferably selected from the group consisting ofSaccharomyces, Kluyveromyces, Candida, Hansenula, Pichia, Komagataellaand Torulopsis, even more preferably the eukaryotic cell is a Pichiacell, most preferably a Pichia pastoris cell.

If the expression vector is intended for being present in a eukaryoticcell, which may be modified or not, it is preferred that the expressionvector comprises features selected from any one of the following: aselection marker; a purification marker; a signal sequence, preferablythe yeast alpha-factor secreting signal sequence, the yeast KILM1 signalpeptide, the yeast PH01 signal peptide, or the yeast SUC2 signalpeptide, more preferably an alpha-factor secreting signal sequence, evenmore preferably the MFalpha pre-pro signal sequence; an origin ofreplication; and/or a nucleotide sequence for targeted (option 1, ofFIG. 3 C) and/or random integration (option 2, FIG. 3 D) into the genomeof a host cell.

Within the meaning of the present invention, the eukaryotic cells thatare used are modified compared to their wildtype cell. This means thatthe modified eukaryotic cell (at least) comprises a modified ssn6-likerelated gene or a modified ssn6-like gene; and/or a modified, preferablyreduced, expression level of SSN6-like related protein or of SSN6-likeprotein, or that an ssn6-like related gene or an ssn6-like gene isdeleted resulting in the complete lack of said SSN6-like related proteinor complete lack of said SSN6-like protein.

The effect of the respective modifications is that the modifiedeukaryotic cell is not able to provide an SSN6-like related protein oran SSN6-like protein that exerts its wildtype function and/or wildtypeactivity, preferably with respect to regulating the LLP promoter; and/orthe amount of SSN6-like related protein or of SSN6-like protein beingpresent in the modified eukaryotic cell differs (preferably in that itis reduced) from the amount of SSN6-like related protein or of SSN6-likeprotein being present in its wildtype form; and/or no SSN6-like relatedprotein or SSN6-like protein is present in the modified cell. This, inturn, has the effect that said modified eukaryotic cell, compared to itswildtype cell, exhibits different (preferably reduced) SSN6-like relatedprotein or SSN6-like protein activity and/or function, preferably withrespect to its activity and/or function in regulating the LLP promoter.

As described elsewhere herein, if the modified eukaryotic cell is notable to provide a proper, sufficiently working SSN6-like related proteinor SSN6-like protein (e.g. in terms of function, activity, and/oramount), as a result the regulatory sequences that are controlled bysaid protein, such as promoters, like the LLP promoter, are not affectedin their function (e.g. are not repressed) any more. This, in turn,results in an overexpression of the protein that is under control ofsaid regulatory polynucleotide sequence.

In other words, proper, sufficiently working SSN6-like related proteinor SSN6-like protein represses the LLP promoter, which means that theLLP promoter exhibits reduced function, when compared to the function ofthe LLP promoter that is not repressed by said proteins. As to the term“reduced”, reference is made to the description elsewhere herein.

Within the meaning of the present invention, the term “overexpression”defines that the expression of a protein (encoded for instance by thegene of interest) in a cell such as a (modified) eukaryotic cell is atlevels greater than normal in a wildtype cell. For instance, in thepresent invention the modified eukaryotic cell can be used in order tooverexpress a gene of interest.

Within the meaning of the present invention, the term “modification”denotes any suitable amendment that is known to a person skilled in theart that can be applied to a nucleotide sequence or gene, respectively,that results in one or more of the following effects (if the modifiednucleotide sequence is expressed in a suitable expression system): theSSN6-like or SSN6-like related protein does not exert its wildtypefunction and/or wildtype activity; the amount of SSN6-like or SSN6-likerelated protein being present is lower as compared to non-modifiedcells; and/or no SSN6-like or SSN6-like related protein is present atall. In an embodiment of the present invention, the nucleotide sequenceof the ssn66-like or ssn66-like related gene, respectively, is modifiedby introduction of a point mutation, e.g. substitution, insertion ordeletion of a single or more nucleotides; by partial or completedeletion; and/or by replacement by a different, e.g. heterologous,nucleotide sequence.

It is also possible that the expression level of the affected SSN6-likeor SSN6-like related protein is modified, for instance by introductionof a point mutation, e.g. substitution, insertion or deletion of asingle or more nucleotides in regulatory polynucleotide sequences of theaffected ssn6-like or ssn6-like related nucleotide sequence or ssn6-likeor ssn6-like related gene, respectively; by partial or complete deletionof regulatory polynucleotide sequences of the affected ssn6-like orssn6-like related nucleotide sequence or ssn6-like or ssn6-like relatedgene, respectively; and/or by replacement of the regulatorypolynucleotide sequences of the affected ssn6-like or ssn6-like relatednucleotide sequence or ssn6 or ssn6-like gene, respectively, bydifferent, e.g. heterologous, nucleotide sequences. The regulatorypolynucleotide sequences are selected from the group as definedelsewhere herein.

In order to assess whether a modification in the meaning of the presentinvention has been carried out on a nucleotide sequence, suitablesequence alignments can be carried out: For instance, the sequence ofthe potentially modified polynucleotide sequence can be aligned with therespective wildtype counterpart. The resulting alignment indicateswhether a modification has been carried out, and if so, which kind ofmodification.

Within the meaning of the present invention, the term “coding sequence”denotes a nucleotide sequence (e.g. heterologous or homologousnucleotide sequence or heterologous or homologous polynucleotide,respectively), such as a nucleotide sequence being comprised in a geneof interest, that encodes an expression product, such as an RNA orpolypeptide, that, when expressed, results in production of the product(e.g. polypeptide (for instance a heterologous polypeptide), such asenzymes, antibodies or fragments thereof, hormones, or protein-antigenspresent in vaccines).

Here, the term “a promoter that is repressed” defines that the promoteris not able to exert its full, natural function and its full, naturalactivity. An example of such a promoter that is repressed in thepresence of SSN6-like or SSN-like related protein is the LLP promoter:It is not yet known which mechanism is underlying said repression,however it is speculated that the repression could be based onalteration of the local chromatin structure, or on interaction with thegeneral transcription machinery (Smith et al., TIBS 25, July 2000,325-330).

Within the meaning of the present invention, as a result of therepression of the promoter, the expression of a gene that is undercontrol of said promoter is influenced to the effect that the respectiveencoded protein is not expressed; and that the amount of said expressedprotein differs from the wildtype amount of said protein.

Within the meaning of the present invention a coding sequence (e.g. of aheterologous polynucleotide) is “under control of” (or the like) atranscriptional or translational control sequence (regulatorypolynucleotide sequence) when the regulatory polynucleotide sequencedirects RNA, preferably mRNA, which then may be spliced (if it containsintrons) and, optionally, translated into a protein encoded by thecoding sequence. In an embodiment of the present invention, a gene (suchas a gene of interest) is under control of the LLP promoter. This meansthat as long as the promoter works as in a wildtype situation (forinstance, is repressed by SSN6-like protein or SSN6-like relatedprotein), a gene that is under control of said promoter is not expressedor expressed at low levels when the cells are cultivated in standardconditions. As soon as the LLP promoter is de-repressed due to thepresence of a modified SSN6-like related protein or SSN6-like protein ordifferent cultivation conditions, a gene that is under control of thispromoter is expressed at high to very high levels (compared to asituation where the promoter is not repressed). Based on the ratiounderlying the present invention, it is likely that a SSN6-like proteinor a SSN6-like related protein might be blocked not only be mutation ofthe ssn6-like gene or the ssn6-like related gene, but also by inhibitorsof ssn6-like gene or protein or by inhibitors of ssn6-like related geneor protein, which also could result in increased activity of theLLP-promoter.

The term “functional” defines that the respective matter exhibits itsnatural function.

The term “selecting” refers to the identification and isolation of arecipient cell that contains the vector of interest. Transformedmicroorganisms, that is, those containing recombinant molecules such asa vector or plasmid, may be selected with a variety of positive and/ornegative selection methods or markers. Details according to suchselection methods and markers are described elsewhere in thisapplication. Negative selection markers include, but are not limited to,genes such as thyA, sacB, gnd, gapC, zwJ, talA, taiB, ppc, gdhA, pgi,Jbp, pykA, cit, acs, edd, icdA, groEL, secA and the like.

In general, the term “expression system” denotes a system that isspecifically designed for the production of a gene product of choice,also referred to herein as protein of interest (POI). In the presentinvention, the expression system comprises a modified ssn6-like relatedgene or a modified ssn6-like gene, or an ssn6-like related gene or anssn6-like gene and a modified regulatory polynucleotide sequence that isinvolved in the regulation of the expression of said genes, to theeffect that said expression system is not able to express an SSN6-likerelated protein or SSN6-like protein, that SSN6-like related protein orSSN6-like protein is expressed that does not exert its wildtype functionand/or wildtype activity, and/or that an amount of SSN6-like relatedprotein or SSN6-like protein is expressed that differs, preferably islower, when compared to the amount of SSN6-like related protein or ofSSN6-like protein that is expressed by said expression system when thecorresponding wildtype polynucleotide sequence is expressed by such anexpression system under the same conditions.

The expression system of the present invention is a eukaryoticexpression system, i.e. an expression system that comprises a eukaryoticcell. In particular, within the meaning of the present invention, saideukaryotic cell is modified as described elsewhere herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is now described in more detail by preferredembodiments and examples, which are however presented for illustrativepurpose only and shall not be understood as limiting the scope of thepresent invention in any way.

Although expression systems for expressing proteins are widely known inprior art, these expression systems often exhibit disadvantages, forinstance with regard to the yield of the expression product, or withregard to the expression system itself.

The present invention is based on the surprising general finding that animproved constitutive eukaryotic expression system can be generated bymodifying an ssn6-like related gene or ssn6-like gene (“ssn6-likerelated/ssn6-like gene”) that is present in a eukaryotic cell, and/or bymodifying the expression level of SSN6-like related protein or SSN6-likeprotein; or by deleting the ssn6-like related gene or ssn6-like gene,respectively to the effect that the resulting modified eukaryotic cellis not able to provide an SSN6-like related protein or an SSN6-likeprotein that exerts its wildtype function and/or wildtype activity inparticular with regard to regulatory sequences such as a promoter thatis regulated by said proteins (such as the LLP promoter); the amount ofSSN6-like related protein or of SSN6-like protein being present in themodified eukaryotic cell differs (preferably is lower) from the amountof SSN6-like related protein or of SSN6-like protein being present inits wildtype form, to the effect that said proteins are not able toexert their wildtype function and/or activity in particular with regardto regulatory sequences such as a promoter that is regulated by saidproteins; and/or no SSN6-like related protein or SSN6-like protein(“SSN6-like related/SSN6-like protein”) is present in the modified cell,again to the effect that said proteins are not able to exert theirwildtype function and/or activity in particular with regard toregulatory sequences such as a promoter that is regulated by saidproteins. Thus, by preventing the SSN6-like related protein or SSN6-likeprotein from exerting its natural (wildtype) function, which functioncomprises the repressive direct and/or indirect interactions withregulatory sequences such as promoters (e.g. the LLP promoter), theexpression of a gene that is under control of said regulatory sequences(e.g. LLP promoter) in a suitable expression system is, at least to acertain degree, not repressed any more. In other other words, therespective gene being under control of said promoter is overexpressedcompared to its wildtype expression.

By applying the principle underlying the present invention, a eukaryoticexpression system is provided, which is for instance improved withregard to expression product yield compared to the yield that isobtained when applying prior art expression systems. Moreover, theinventive expression system is improved with regard to safety aspects:Contrary to certain prior art eukaryotic expression systems, the presenteukaryotic expression system is not based on the commonly used AOX(alcohol oxidase) promoters, which are tightly regulated by methanol.The presence of flammable and toxic methanol represents a major securityrisk, and also imposes a risk on alcohol-sensitive expression productsor host cells. Especially in large scale fermentation of recombinantproteins the use of methanol is a serious safety and environmental risk.Alternative eukaryotic expression systems that use promoters that arenot regulated by methanol are for instance based on the constitutive GAP(glyceraldehyde-3-phosphate dehydrogenase) promoter. However, theresulting yield for instance when applying a GAP-based expression systemleaves much to be desired.

Within the meaning of the present invention it has been surprisinglyfound that the modification of an ssn6-like related gene or of anssn6-like gene and/or the modification of the expression level ofSSN6-like related protein or SSN6-like protein that is present in aeukaryotic cell to the effect that the resulting modified eukaryoticcell is not able to exert the wildtype function of SSN6-like relatedprotein or SSN6-like protein with regard to a regulatory sequence thatis regulated by said SSN6-like related protein or SSN6-like protein,such as a promoter (e.g. the LLP promoter), results in an enhancedexpression of the gene that is under control of said regulatorysequence.

Thus, the present invention provides a modified eukaryotic cell, whichis modified compared to its wildtype cell at least in that it comprises

-   -   a modified ssn6-like related gene or a modified ssn6-like gene,        and/or    -   a modified expression level of SSN6-like related protein or of        SSN6-like protein, or    -   in that    -   an ssn6-like related gene or an ssn6-like gene is deleted,

respectively to the effect that

-   -   the modified eukaryotic cell is not able to provide an SSN6-like        related protein or an SSN6-like protein that exerts its wildtype        function and/or wildtype activity,    -   the amount of SSN6-like related protein or of SSN6-like protein        being present in the modified eukaryotic cell differs from the        amount of SSN6-like related protein or of SSN6-like protein        being present in its wildtype form, and/or    -   no SSN6-like related protein or SSN6-like protein is present in        the modified cell.

In accordance with the ratio underlying the present invention, theresulting modified eukaryotic cell, when compared to its wildtype cell,exhibits different SSN6-like related protein or SSN6-like proteinactivity and/or function, preferably with respect to its activity and/orfunction in regulating the expression of a gene.

Whenever the term “wildtype function” is used in connection withSSN6-like related protein or SSN6-like protein, or the respective genesencoding said proteins, the function with regard to a regulatorysequence that is regulated by SSN6-like related protein or SSN6-likeprotein, such as a promoter (preferably the LLP promoter), is referredto.

As it is known to a person skilled in the art, the expression of a geneis, amongst others, regulated by a complex interplay of various factorsthat contribute to the regulation of the expression of (a) certaingene(s), such as promoters, enhancers, terminators, silencers,transcription factors, IRES-sequences, ribosome-binding sites, andsequences stabilizing or destabilizing the mRNA by secondary structuresor enhancers.

Thus, in a preferred embodiment, the resulting modified eukaryotic cellexhibits different SSN6-like related protein or SSN6-like proteinactivity and/or function with regard to any promoter that is regulatedby said proteins, preferably the LLP promoter. Hence, it is a preferredembodiment of the present invention that the modified eukaryotic cell,when compared to its wildtype cell, exhibits different SSN6-like relatedprotein or SSN6-like protein activity and/or function with regard toregulating the expression of genes that are under control of the LLPpromoter. The activity and/or function of said proteins (SSN6-likerelated and SSN6-like) results in that the genes that are under controlof said promoter (LLP-promoter) are overexpressed.

The different activity and/or function of the SSN6-like related proteinor SSN6-like protein can be any different activity and/or function thatresults in an overexpression of the respective gene that is undercontrol of the affected regulatory sequence. For example an SSN6-likerelated protein or SSN6-like protein might be altered in its activityand/or function by “gain of function” or “loss of function”modifications. If several copies of different modified versions orwild-type SSN6-like related protein or SSN6-like protein are presentwithin a cell at the same time, certain SSN6-like related proteins orSSN6-like proteins might be dominant regarding their activity and/orfunction. “Loss-of-function” mutants comprise a mutation that results inreduced or abolished protein function. “Gain-of-function” mutantscomprise a mutation that results in an abnormal activity on a protein.Preferably, it is a reduced activity and/or function of the SSN6-likerelated protein or of the SSN6-like protein. It can also be that thereis no SSN6-like related protein or SSN6-like protein at all present inthe modified eukaryotic cell, or that there is no SSN6-like relatedprotein or SSN6-like protein within the detection limit of commonly knowmethods such as for example RT-PCR, qPCR or other PCR-relatedtechniques, western blotting, ELISA, or other immunological detectionassays, reporter gene assays, mass spectrometric detection methods, chipassays detecting proteins or nucleic acids, etc.

In order to arrive at a modified eukaryotic cell that exhibitsdifferent, preferably reduced, more preferably essentially no, SSN6-likerelated protein or SSN6-like protein function and/or activity, thessn6-like related gene or ssn6-like gene can be modified. Thismodification can for instance have taken place in a coding sequenceencoding the respective protein, and/or in a respective regulatorysequence. A regulatory sequence that can be modified is for instance apromoter, enhancer, terminator, silencer, IRES-sequence,ribosome-binding site, and sequence stabilizing or destabilizing themRNA by secondary structures or enhancer.

According to the present invention, the above modifications on theregulatory and/or coding regions of the genes encoding SSN6-like relatedprotein and/or SSN6-like protein have an effect on the expression levelof the SSN6-like related protein or SSN6-like protein in that themodified eukaryotic cell is not able to express said proteins, or thatthe amount of said proteins is different, preferably reduced, whencompared with its wildtype form. The above modifications can also havean effect on the function and/or activity of SSN6-like related proteinor SSN6-like protein in that said proteins do not exert their wildtypefunction and/or activity. It is also possible to arrive at a modifiedeukaryotic cell exhibiting impaired SSN6-like related/SSN6-like proteinas defined above by impairing the transcription or translation of thegene encoding SSN6-like related/SSN6-like protein. Transcription andtranslation can be impaired by any suitable method that is known to aperson skilled in the art, for instance translation can be impaired byhybridizing a complementary sequence or a partial sequence thereof tothe mRNA transcript. The partial sequence of the complementary sequenceis for example selected from siRNA, anti-sense RNA, ribozyme, andtriplex RNA or DNA, etc.

Irrespective of whether the regulatory and/or coding regions of thessn6-like related/ssn6-like gene is modified, or whether thetranscription and/or translation of the gene encoding SSN6-likerelated/SSN6-like protein is impaired, as a result, the modifiedeukaryotic cell is not able to provide an SSN6-like related protein oran SSN6-like protein that exerts its wildtype function and/or wildtypeactivity, the amount of SSN6-like related protein or of SSN6-likeprotein being present in the modified eukaryotic cell differs from theamount of SSN6-like related protein or of SSN6-like protein beingpresent in its wildtype form, and/or no SSN6-like related protein orSSN6-like protein is present in the modified cell, as it is disclosedelsewhere herein.

The ssn6-like related/ssn6-like gene and/or the expression level of theSSN6-like related/SSN6-like protein can be modified by any suitablemethod that is known to a person skilled in the art. For instance, thessn6-like related/ssn6-like gene and/or the expression level of theSSN6-like related/SSN6-like protein is modified by

-   -   introduction of one or more point mutations, e.g. substitution,        insertion or deletion of a single or more nucleotides in the        polynucleotide sequence of the ssn6-like related gene or of the        ssn6-like gene,    -   partial or complete deletion of the polynucleotide sequence of        the ssn6-like related gene or of the ssn6-like gene, and/or    -   partial or complete replacement of the polynucleotide sequence        of the ssn6-like related gene or of the ssn6-like gene by a        different, e.g. heterologous, nucleotide sequence.

In a preferred embodiment, the total activity, function, preferably generegulatory function, and/or amount of SSN6-like related protein orSSN6-like protein of the modified eukaryotic cell is at most 20%,preferably at most 15%, more preferably at most 10%, even morepreferably at most 5% of the activity, function and/or amount ofSSN6-like related protein or SSN6-like protein of its wildtype form, andmost preferably there is no activity and/or no function of the SSN6-likerelated protein or SSN6-like protein of the modified eukaryotic cell atall.

Within the meaning of the present invention, the term “total activity,function, and/or amount of SSN6-like related protein or SSN6-likeprotein of the modified eukaryotic cell” defines the overall activity,function, and/or amount of SSN6-like related protein or SSN6-likeprotein of a sample of modified eukaryotic cells. For instance, in orderto assess whether the activity, function, and/or amount of SSN6-likerelated protein or SSN6-like protein of the modified eukaryotic cellsdiffers from the corresponding feature of the wildtype cells, the totalamount of SSN6-like protein being present in a sample of a certainnumber of wildtype cells is compared with the total amount of SSN6-likeprotein being present in a sample of essentially the same number ofmodified eukaryotic cells.

Within the meaning of the present invention, the modified eukaryoticcell can be any suitable cell, i.e. any suitable eukaryotic cell thatcomprises, in its wildtype form, a polynucleotide sequence encodingSSN6-like related/SSN6-like protein. In a preferred embodiment, saidsuitable cell additionally comprises a nucleotide sequence representingthe LLP promoter. In order to determine whether a eukaryotic cellcomprises the above defined sequences, any suitable method that is knownto a person skilled in the art can be used, for instance techniques suchas northern blotting, real time PCR, reverse transcriptase quantitativePCR (RT-qPCR), or microarray hybridization experiments. Microarrayhybridization experiments can be used to quantify the expression ofhundreds to thousands of genes at the same time. Suitable protocols canfor instance be found in Sambrook et al.

In a preferred embodiment of the present invention, the modifiedeukaryotic cell is a fungal cell, preferably a yeast cell, morepreferably selected from the group consisting of Saccharomyces species(e.g., Saccharomyces cerevisiae), Kluyveromyces species (e.g.,Kluyveromyces lactis), Torulaspora species, Yarrowia species (e.g.,Yarrowia lipolitica), Schizosaccharomyces species (e.g.,Schizosaccharomyces pombe), Pichia species (e.g., Pichia pastoris orPichia methanolica), Hansenula species (e.g., Hansenula polymorpha),Torulopsis species, Komagataella species, Candida species (e.g., Candidaboidinii), and Karwinskia species, even more preferably the eukaryoticcell is a Pichia cell, most preferably a Pichia pastoris cell accordingto current classification.

In a further preferred embodiment, the modified eukaryotic cell is a P.Pastoris cell, preferably a cell of a strain selected from the groupconsisting of NRRL Y-11430, CBS704, GS115, and KM71.

As disclosed herein, the modified eukaryotic cell according to thepresent invention can comprise a modified ssn6-like related gene or amodified ssn6-like gene. Thus, in a further embodiment, the modifiedeukaryotic cell comprises a polynucleotide sequence which represents amodification of SEQ ID NO: 1.

As explained elsewhere herein, it has been found that a modification ofssn6-like related/ssn6-like gene according to the present invention,i.e. a modification that results in a modified expression of SSN6-likerelated/SSN6-like protein, results in an overexpression of genes thatare under control of a promoter that is regulated by SSN6-likerelated/SSN6-like protein, such as the LLP promoter. Thus, the presentinvention also refers to a polynucleotide sequence comprising

-   -   a modified ssn6-like related gene or a modified ssn6-like gene        comprising a coding sequence and a regulatory sequence,

wherein, if said polynucleotide sequence is expressed in a suitableexpression system,

-   -   essentially no SSN6-like related protein or SSN6-like protein is        expressed, or    -   SSN6-like related protein or SSN6-like protein is expressed that        does not exert its wildtype function and/or wildtype activity,        preferably with respect to its activity and/or function in        regulating gene expression, more preferably in regulating the        expression of genes that are under control of the LLP promoter,        and/or    -   an amount of SSN6-like related protein or SSN6-like protein is        expressed that differs from the amount of SSN6-like related        protein or of SSN6-like protein that is expressed when the        corresponding wildtype polynucleotide sequence is expressed        under comparable or the same conditions.

In a preferred embodiment, the amount of SSN6-like related protein orSSN6-like protein that is expressed differs from the amount of SSN6-likerelated protein or of SSN6-like protein that is expressed when thecorresponding wildtype polynucleotide sequence is expressed undercomparable or the same conditions in that it is reduced.

In a further embodiment, the above polynucleotide consists of saidmodified ssn6-like/ssn6 gene.

In a preferred embodiment, the amount of SSN6-like related/SSN6-likeprotein is reduced, or there is no SSN6-like related protein orSSN6-like protein present at all, compared to the amount of SSN6-likerelated/SSN6-like protein that is expressed when the correspondingwildtype polynucleotide sequence is expressed under comparable or thesame conditions.

The modifications can be carried out in the regulatory sequence(regulatory region) and/or in the coding sequence (coding region) of thessn6-like related/ssn6-like gene. The regulatory sequence can be anyregulatory sequence, preferably the regulatory sequence is selected fromthe group comprising or consisting of promoters, enhancers, terminators,silencers, IRES-sequences, ribosome-binding sites, and sequencesstabilizing or destabilizing the mRNA by secondary structures, morepreferably said regulatory sequence is selected from the groupconsisting of promoters, enhancers, and terminators.

The ssn6-like related/ssn6-like gene has been modified as disclosedelsewhere herein. In a preferred embodiment, the polynucleotidecomprises, preferably consists of, modifications of SEQ ID NO: 1. Anexample of a modified ssn6-like gene is depicted in FIG. 1 G (SEQ ID NO:7). Thus, in a further preferred embodiment, the polynucleotideaccording to the present invention comprises, preferably consists of,SEQ ID NO: 7.

As it is known to a person skilled in the art, it is possible topropagate and/or express a certain nucleotide sequence by inserting thissequence into a vector, which is then, in turn, introduced in a hostcell such as a bacterium, with this bacterium preferably beingEscherichia coli (E. coli) or a eukaryotic cell for instance asdisclosed elsewhere herein. Thus, the present invention also refers to avector comprising the polynucleotide sequence comprising the modifiedssn6-like related/ssn6-like gene, as well as to a host cell comprisingsaid vector or parts of said vector.

Depending on the respective intended use, said vector contains specificstructural elements. If it is for instance intended to propagate thenucleotide sequence, then the corresponding vector has to contain anori.

The present invention also refers to a polypeptide encoded by thepolynucleotide sequence according to the present invention. If thepolynucleotide sequence according to the present invention, comprising amodified ssn6-like related/ssn6-like gene as disclosed above, isexpressed in a suitable expression system, the expression of a geneproduct, e.g. of a polypeptide or protein, depends on the modificationsthat have been carried out. For instance, if the modification results ina shift of the complete reading frame, in all likelihood no gene productcan be expressed. It can however also be that the modification onlyresults in a truncated (“shortened”) nucleotide sequence, which in turnresults in the expression of a truncated gene product which may alsocontain a short heterologous amino acid sequence at its C-terminus whichis coded by the inserted heterologous nucleotide sequence (see FIG. 2 B,SEQ ID NO: 9, with 7 heterologous C-terminal amino acids (EWYLQLR,underlined in FIG. 2 B; SEQ ID NO: 139), as compared to the non-mutatedsequence in FIG. 2 A, SEQ ID NO: 8).

In the present invention it has been surprisingly found that bypartially or completely inhibiting the wildtype function and/or activityof SSN6-like related/SSN6-like protein, or by effecting that SSN6-likerelated/SSN6-like protein is essentially not present, respectively in acell that in its wildtype form comprises a gene encoding said protein, agene being under the control of a promoter that is repressed in thepresence of SSN6-like related/SSN6-like protein can be overexpressed ina suitable expression system.

Thus, the present invention further refers to an expression vectorcomprising a promoter, wherein said promoter is characterized in that itis repressed in the presence of SSN6-like protein or SSN6-like relatedprotein if said expression vector is introduced into a suitableexpression system, e.g. into a eukaryotic cell as defined elsewhereherein. By using this expression vector, a gene under control of apromoter that is repressed in the presence of SSN6-likerelated/SSN6-like protein can be overexpressed in a suitable expressionsystem. In order to determine whether a promoter is repressible bySSN6-like related/SSN6-like protein, any suitable method that is knownto a person skilled in the art can be used. For instance, expressionvectors can be designed that comprise the promoter to be tested, and anindicator gene (also called reporter-gene) such as luciferase that isunder control of the promoter to be tested. If, in the presence ofSSN6-like related/SSN6-like protein, the expression of the gene productis reduced, then the candidate promoter is repressed in the presence ofSSN6-like related/SSN6-like. Determination of the reduced candidatepromoter activity for example can be measured by quantifying theindicator gene product or its activity, for example luciferase, bymethods known in the art. There are a number of assays know in the art,which can be used to determine promoter-activity or measure interactionof proteins with promoter-sequences and which thereby are suitable totest whether the activity of a certain promoter is influenced by anSSN6-like or SSN6-like related protein. Examples of such assays arereporter gene assays, electrophoretic mobility shift assays (EMSA, gelshift assays), double-stranded DNA pull-down assay, chromatinimmune-precipitation assays, and DNase foot printing assays, etc.

In a preferred embodiment of the present invention, the expressionvector comprises one or more gene(s) of interest that are under controlof said promoter, i.e. the promoter that is repressed in the presence ofSSN6-like related/SSN6-like protein. By applying suitable protocols thatare known to a skilled person, such as a protocol as disclosed above, aperson skilled in the art is able to assess whether a promoter isrepressed in the presence of SSN6-like related/SSN6-like protein, ornot. In a preferred embodiment of the present invention, the promoterbeing comprised in the expression vector and controlling the gene(s) ofinterest is an LLP promoter or a modified version thereof. Preferably,the LLP promoter comprises, preferably consists of, the polynucleotidesequence depicted in SEQ ID NO: 2 or 12. The term “modified version” ofthe LLP promoter defines that said modified LLP promoter still exhibitsthe function of an LLP promoter in terms of regulating the expression ofthe gene that is under its control. It is further possible that thepromoter is an LLP promoter, and that LLP is encoded or is not encodedby the polynucleotide sequence of the expression vector. In either case,and also in case the promoter is not LLP promoter but a differentpromoter that is repressed in the presence of SSN6-likerelated/SSN6-like protein, the gene of interest preferably is selectedfrom the group consisting of genes encoding enzymes, antibodies orfragments thereof, hormones, structural proteins, and protein-antigensbeing present in vaccines.

Preferably, the expression vector further comprises features selectedfrom any one of the following:

-   -   (i) a selection marker;    -   (ii) a purification marker;    -   (iii) a signal sequence, preferably an alpha-factor secreting        signal sequence, more preferably the MFalpha pre-pro signal        sequence, even more preferably a signal sequence comprising or        consisting of, preferably consisting of, SEQ ID NO: 14 or SEQ ID        NO: 21;    -   (iv) an origin of replication; and/or    -   (v) a nucleotide sequence for targeted and/or random integration        into the genome of a host cell.

The expression vector can be comprised in a host cell as describedherein, preferably in a bacterium such as E. coli, for instance forpropagation purposes.

The expression vector can also be introduced into the modifiedeukaryotic cell according to the present invention, resulting in theintroduction of the expression vector's expression cassette into themodified eukaryotic cell's genome. The introduction of the expressionvector into the host cell can be carried out according to any suitablemethod that is known to a skilled person, for instance byelectroporation. In addition, transient expression, i.e. the expressionvector is not integrated into the genome, can also be considered.

The present invention also refers to a modified eukaryotic cellcomprising the expression vector as defined herein.

The introduction of the expression cassette of the expression vectorinto the modified eukaryotic cell is also denoted as “recombination” andcan be a targeted, i.e. homologous, recombination, or a randomrecombination. If a random recombination takes place, the expressioncassette integrates in such a way into the genome of the modifiedeukaryotic cell that the nucleotide sequence encoding LLP proteinpresent in the modified eukaryotic cell is not affected. In other words,the modified eukaryotic cell expresses not only the gene of interest,but also the LLP protein. In this case, the amount of LLP protein beingpresent in the modified cell, the supernatant of the modified cell or insubfractions thereof can be used as an indicator of the purity of theexpression product of the gene of interest, i.e. the protein ofinterest, for example during purification of the protein of interest.This situation, where the LLP protein is expressed in addition to thegene(s) of interest, is referred to as “Option 2” (FIG. 3 D).

Thus, the present invention also refers to a eukaryotic cell comprising

-   -   a modified ssn6-like related gene or a modified ssn6-like gene,        wherein said gene has inserted a foreign nucleotide sequence, to        the effect that    -   (i) the eukaryotic cell is not able to provide an SSN6-like        related protein or an SSN6-like protein that exerts its wildtype        function and/or wildtype activity, preferably with respect to        its activity and/or function in regulating the LLP promoter,    -   (ii) the amount of SSN6-like related protein or of SSN6-like        protein being present in the eukaryotic cell differs from the        amount of SSN6-like related protein or of SSN6-like protein        being present in its wildtype form, preferably the amount of the        SSN6-like related protein or of the SSN6-like protein is        reduced, and/or    -   (iii) no SSN6-like related protein or SSN6-like protein is        present in the eukaryotic cell,    -   a gene of interest under control of the LLP promoter, and    -   an llp gene,

preferably wherein said eukaryotic cell exhibits reduced SSN6-likerelated protein or SSN6-like protein activity and/or function, or noSSN6-like related protein or SSN6-like protein activity and/or functionat all.

If a targeted, i.e. homologous, recombination takes place, and ifadditionally the promoter of the expression cassette is an LLP promoteror a modification thereof, then the expression cassette is inserted insuch a way that it replaces (partially or completely) the nucleotidesequence of the gene encoding the LLP protein. In this case, there is noexpression of LLP. This situation, where only the gene(s) of interest,but no LLP protein, is expressed, is referred to as “Option 1”.

Thus, the present invention also refers to a eukaryotic cell comprising

-   -   a modified ssn6-like related gene or a modified ssn6-like gene,        wherein said gene has inserted a foreign nucleotide sequence, to        the effect that    -   (i) the eukaryotic cell is not able to provide an SSN6-like        related protein or an SSN6-like protein that exerts its wildtype        function and/or wildtype activity, preferably with respect to        its activity and/or function in regulating the LLP promoter,    -   (ii) the amount of SSN6-like related protein or of SSN6-like        protein being present in the eukaryotic cell differs from the        amount of SSN6-like related protein or of SSN6-like protein        being present in its wildtype form, preferably the amount of the        SSN6-like related protein or of the SSN6-like protein is        reduced, and/or    -   (iii) no SSN6-like related protein or SSN6-like protein is        present in the eukaryotic cell,

and

-   -   a gene of interest that replaces a part or all of the coding        region of the gene encoding the LLP protein, to the effect that        essentially no LLP protein is present in the eukaryotic cell,        and that is under control of the LLP promoter,

preferably wherein said eukaryotic cell exhibits reduced SSN6-likerelated protein or SSN6-like protein activity and/or function, or noSSN6-like related protein or SSN6-like-protein activity and/or functionat all.

The present invention further refers to an expression system, comprising

-   -   a) the modified eukaryotic cell as defined herein;    -   b) the expression vector as defined herein, wherein said        expression vector can also be present in linearized form and/or        at least parts of the vector being integrated into the genome of        the modified eukaryotic cell.

In a further preferred embodiment of the present invention, the modifiedeukaryotic cell additionally comprises a promoter, wherein said promoteris repressed in the presence of SSN6 protein or SSN6-like protein.Preferably, said promoter is an LLP promoter, preferably an LLP promotercomprising, preferably consisting of, SEQ ID NO: 2 or 12, or modifiedversions thereof, said modified versions being characterized in thatthey still exhibit essentially the same promoter function as a wildtypeLLP promoter.

In a further embodiment, the modified eukaryotic cell additionallycomprises (a) gene(s) of interest, preferably selected from the groupconsisting of genes encoding enzymes, antibodies or fragments thereof,hormones, structural proteins, and protein-antigens being present invaccines, wherein said gene(s) of interest is (are) under control of apromoter being repressed in the presence of SSN6-like protein orSSN6-like related protein. Preferably, this promoter is an LLP promoter,preferably an LLP promoter comprising, preferably consisting of, SEQ IDNO: 2 or 12, or modified versions thereof, said modified versions beingcharacterized in that they still exhibit essentially the same promoterfunction as a wildtype promoter.

As already explained above, it is possible that in said expressionsystem the LLP gene is not expressed in addition to the gene(s) ofinterest (option 1), and it is also possible that the LLP gene isexpressed in addition to the gene(s) of interest (option 2). In thefirst case (option 1), the LLP promoter only controls the expression of(a) gene(s) of interest, and in the latter case (option 2), the LLPpromoter controls the expression of (a) gene(s) of interest (which aredifferent from genes encoding LLP), and LLP.

The present invention further refers to the use of the modifiedeukaryotic cell, the modified polynucleotide sequence, the expressionvector, the host cell, or an expression system as defined herein in amethod of expressing, preferably overexpressing, gene(s) of interest.

As disclosed above, the amount of LLP protein being present in acomposition comprising the protein of interest is an indicator of thepurity of said composition regarding said protein of interest. Thus, thepresent invention also refers to a method for determining the purity ofa composition comprising the expression product of a gene of interest,comprising the following steps:

-   -   (a) expressing gene(s) of interest by using the modified        eukaryotic cell and the expression vector, respectively        according to the present invention, wherein        -   (a1) the modified eukaryotic cell comprises a gene encoding            LLP protein under control of an LLP promoter, and wherein        -   (a2) the expression vector comprises one or more gene(s) of            interest under control of an LLP promoter or a modified LLP            promoter, wherein said gene of interest does not encode LLP,    -   thereby obtaining a composition comprising the expression        product of the gene(s) of interest, i.e. the protein(s) of        interest, and the LLP protein;    -   (b) determining the amount of the expression product of the        gene(s) of interest, i.e. the amount of the protein(s) of        interest, and the amount of LLP protein being present in the        composition obtained in step (a), wherein the amount of LLP        protein compared to the amount of expression product of the        gene(s) of interest, i.e. of the protein(s) of interest, is        indicative for the purity of the composition obtained in step        (a); and, optionally,    -   (c) subjecting the composition of step (a) to one or more        downstream purification step(s), followed by step b), i.e.        determining the amount of the expression product of the gene(s)        of interest, i.e. the protein(s) of interest, and the amount of        LLP protein being present in the composition obtained after        having carried out said downstream purification step.

In order to obtain a composition that comprises LLP, in addition to thegene(s) of interest, the integration of the expression cassette of theexpression vector represents a random integration into the genome of themodified eukaryotic cell.

The composition comprising the protein(s) of interest and the LLPprotein can be any composition that arises in the course of the processof producing the protein(s) of interest. Accordingly, such a compositioncan for instance be a supernatant of the cell culture used in expressingthe protein(s) of interest

The present invention further refers to a method of expressing one ormore gene(s) of interest in a eukaryotic cell comprising an ssn6-likerelated gene or an ssn6-like gene, wherein the translation of the mRNAtranscript of the ssn6-like related gene or the ssn6-like gene isprevented by hybridizing a complementary sequence or a partial sequencethereof to the mRNA transcript. In a preferred embodiment, the partialsequence of the complementary sequence is a siRNA, anti-sense RNA, aribozyme, or triplex RNA or DNA.

In a further embodiment, the present invention refers to a method ofexpressing one or more gene(s) of interest in a eukaryotic cellcomprising an SSN6-like expression cassette or SSN6-like relatedexpression cassette, wherein the SSN6-like protein or the SSN6-likerelated protein in said eukaryotic cell is modified or inhibited in itsfunction and/or activity. Preferably, the SSN6-like protein or SSN6-likerelated protein of said eukaryotic cell is modified or inhibited in itsfunction and/or activity in regulating the LLP promoter, more preferablythe SSN6-like protein or SSN6-like related protein exhibits reducedSSN6-like related protein or SSN6-like protein activity and/or function,or no SSN6-like related protein or SSN6-like protein activity and/orfunction.

DESCRIPTION OF THE FIGURES

FIG. 1: Polynucleotide sequences

FIG. 1 shows various polynucleotide sequences.

FIG. 1 A shows the nucleotide sequence of the coding region of thessn6-like gene of P. Pastoris (SEQ ID. NO: 1).

FIG. 1 B shows the nucleotide sequence of the LLP promoter of P.pastoris, 605 bp 3′ from the ATG start codon of LLP (SEQ ID NO: 2).

FIG. 1 C shows the nucleotide sequence of the signal sequence of the LLPprotein of P. Pastoris (SEQ ID NO: 3).

FIG. 1 D shows the nucleotide sequence of the terminator sequence of llpgene of P. Pastoris (SEQ ID NO: 4).

FIG. 1 E shows the nucleotide sequence of the SSN6-like promoter of P.Pastoris, 1000 bp 3′ from the ATG start codon of SSN6-like (SEQ ID NO:5).

FIG. 1 F shows the nucleotide sequence of the terminator sequence ofssn6-like gene of P. Pastoris (SEQ ID NO: 6).

FIG. 1 G shows the nucleotide sequence of the SSN6-like modified DNA ofP. Pastoris (from the ATG start to the TAA stop codon) as present in P.Pastoris strain SSS1. This results in a ssn6-like coding region with aninternal insertation of a heterologous nucleotide sequence, whichdisrupts the coding sequence of ssn6-like coding sequence (SEQ ID NO:7).

FIG. 1 H shows the nucleotide sequence of the Kozak start sequence (SEQID NO: 11).

FIG. 1 I shows the nucleotide sequence of the LLP promoter, 1000 bp 3′from the ATG start codon of LLP (SEQ ID NO: 12).

FIG. 1 J shows the amino acid sequence of the SSN6-like protein (SEQ IDNO: 13)

FIG. 1 K shows MFalpha pre-pro signal sequence: FIG. 1 K (A) shows saidsignal sequence without EAEA repeat (SEQ ID NO: 14), and FIG. 1 K (B)shows said signal sequence with EAEA repeat (SEQ ID NO: 21).

FIG. 1 L shows the codon optimized DNA sequence of encoding a singlechain antibody fragment DLX521 (SEQ ID NO: 15).

FIG. 1 M shows the coding region of the DNA sequence of the llp-geneincluding the signal sequence (SEQ ID NO: 16).

FIG. 1 N shows the codon optimized nucleotide sequence of the humanGrowth Hormone (hGH) as used in the examples of this application (SEQ IDNO: 17)

FIG. 1 O shows the nucleotide sequence of the human serum albumin (HSA)as used in the examples of this application (SEQ ID NO: 18).

FIG. 1 P shows the nucleotide sequence of the penicillin V amidase (PVA)as used in the examples of this application (SEQ ID NO: 19).

FIG. 1 Q shows the nucleotide sequence of the vector pGAPk used forgenerating the SSS1 yeast cell line (plasmid without a GOI and with aGeneticin resistance marker) (SEQ ID NO: 20)

FIG. 1 R shows the sequence of a pLLP vector containing a Geneticinresistance marker (pLLPk) corresponding to SEQ ID NO: 22.

FIG. 1 S shows SEQ ID NO: 23, which is part of the nucleotide sequenceof chromosome 1 of the genomic sequence of yeast strain YJK_PVA_021after random integration of a PVA- and a Zeocin-expression cassette intothe coding region of ssn6-like (reverse-complement sequence ofssn6-like) at position 807,480 of chromosome 1 of the reference strainPichia pastoris CBS 7435. The ssn6-like sequence is underlined, thestart codon is shown in bold and double-underlined(ATG→reverse-complement→CAT). Shown is the interrupted ssn6-like codingsequence including 10 nucleotides flanking 5′ and 3′ to the ssn6-likecoding sequence. The sequence shown is part of the Illumina-sequencedgenome of strain YJK_PVA_021 (SEQ ID NOs: 67-115) and the sequence ofSEQ ID NO: 23 is part of the genomic sequence SEQ ID NO: 103 obtained byIllumina Inc. Sequences of oligo2395 (reverse-complement sequence ofoligo2395) and oligo2398 are labeled in grey.

FIG. 1 T shows the LLP signal sequence fused to the 3′ end of HSA codingsequence replacing the native HSA-signal sequence resulting in thesequence according to SEQ ID NO: 121.

FIG. 1 U shows SEQ ID NO: 129, which is the shortened LLP-promotersequence delta29 corresponding to a 576 bases length of the shortenedLLP-promoter.

FIG. 1 V shows SEQ ID NO: 130, which is the shortened LLP-promotersequence delta93 corresponding to a 512 bases length of the shortenedLLP-promoter.

FIG. 1 W shows SEQ ID NO: 131, which is the shortened LLP-promotersequence delta133 corresponding to a 472 bases length of the shortenedLLP-promoter.

FIG. 1 X shows SEQ ID NO: 132, which is the shortened LLP-promotersequence delta201 corresponding to a 404 bases length of the shortenedLLP-promoter.

FIG. 1 Y shows SEQ ID NO: 133, which is the shortened LLP-promotersequence delta233 corresponding to a 372 bases length of the shortenedLLP-promoter.

FIG. 1 Z shows SEQ ID NO: 134, which is the shortened LLP-promotersequence delta300 corresponding to a 305 bases length of the shortenedLLP-promoter.

FIG. 2: Amino acid sequences

FIG. 2 shows various amino acid sequences.

FIG. 2 A shows the amino acid sequence of the SSN6-like protein of P.Pastoris (SEQ ID NO: 8).

FIG. 2 B shows the amino acid sequence of the SSN6-like modified proteinof P. Pastoris YJK_PVA_021 (SEQ ID NO: 9). The underlined 7 amino acidsrepresent heterologous, non-SSN6-like amino acids originating from thevector used to inactivate the SSN6-like protein.

FIG. 2 C shows the amino acid sequence of the LLP protein including LLPsignal sequence (SEQ ID NO: 10).

FIG. 2 D shows the amino acid sequence of the LLP signal sequence of p.pastoris (SEQ ID NO: 140).

FIG. 3: Mechanism of super-secretor Pichia pastoris (SSS1)

wt=wild-type, ssn6-like=ssn6-like gene, LLP=LLP coding sequence,PI=LLP-promoter, LLP-prot.=LLP-protein, Pg=GAP-promoter, AR=antibioticresistance, GOI=coding sequence of gene of interest, GOI-prot.=gene ofinterest protein

FIG. 3 A illustrates a polynucleotide sequence which is a wt-strain(wild-type strain, NRRL Y-11430) which contains an intact ssn6-like genewhich according to our interpretation suppresses the promoter (PI) ofthe lectine like protein (LLP). The ssn6-like gene is on the reversestrand of chromosome 1 (coding sequence is from 806379-808244) and theLLP gene is on the forward strand of chromosome 1 (coding sequence isfrom 2492530 to 2493945).

FIG. 3 B illustrates an expression construct with a randomly integratedPg/AR sequence into the coding sequence of ssn6-like gene of strain NRRLY-11430 was obtained. The resulting strain contains a disruptedssn6-like gene (inact. ssn6-like). According to our interpretation,disruption of the ssn6-like resulting in inact. ssn6-like removes thesuppressing effect of ssn6-like on the PI-promoter, thereby activatingof the PI-promoter finally resulting in high expression of LLP-proteinor of other GOIs (Gene Of Interest) under control of a LLP-promoter.

FIG. 3 C shows a gene of interest (GOI) which can be introduced into theyeast strain of Fig. B by either homologous recombination therebyreplacing the coding region of the LLP-gene by the coding sequence ofthe GOI (FIG. 3C, =Option 1), and resulting in expression of the GOI(GOI-protein), whereas no LLP-protein is produced.

FIG. 3 D alternatively shows a gene of interest (GOI) fused to theLLP-promoter (PI, GOI) which can be introduced into the yeast strain ofFig. B by random recombination leaving the LLP intact and inserting theGOI under control of the LLP-promoter (PI) into a random position of theyeast genome. This results in concomit expression of the GOI (GOI-prot.)and of LLP (LLP-prot.).

FIG. 4: SDS-PAGE analysis

A codon optimized (done by DNA2.0 Inc., Menelo Park, USA) DNA sequenceencoding the model protein DLX521 (an scFv=single chain variableFragment of an antibody) was inserted into a plasmid with theGAP-promoter (pGAP=pJ905 from DNA2.0 Inc.) between restriction sitesEcoRI and NotI, and a plasmid with the LLP-promoter (pLLP) betweenrestriction sites EcoRV and NotI. The pGAP plasmid was linearized withSwaI and transformed in NRRL Y-11430 and the pLLP plasmid was linearizedwith AvrII and transformed in SSS1. For the pGAP plasmid, transformantswere PCR screened and 8 clones with a positive PCR signal were randomlypicked and used for glycerol stock preparation. For the pLLP plasmid,transformants were PCR screened and 9 clones with a positive PCR signalwere randomly picked and used for glycerol stock preparation. Theglycerol stocks of the 8 pGAP and 9 pLLP strains were subjected toexpression studies at microtiter plate scale (25° C., 350 rpm, 70 h).The OD (optical density) at 600 nm was comparable at harvest (OD wasdetermined at harvest to check for biomass variability). Subsequently,15 μL of the samples (supernatant mixed with NuPAGE® LDS sample bufferincl. 2-mercaptoethanol according to manufacturers (Invitrogen/LifeTechnologies) instructions) were loaded onto the gel.

The SDS-polyacrylamide gel (SDS-PAGE) system used was the Novex NuPAGE®Bis-Tris 4-12% gradient gel (Invitrogen/Life Technologies) using theMES-buffer system (Invitrogen/Life Technologieis). The protein molecularweight marker (M) used was from AppliChem GmbH, Darmstadt, Germany. Theused Protein Marker VI, prestained (AppliChem), shows the indicatedapproximate molecular weights in kilo Dalton (kDa) of the markerproteins if separated in a 10% SDS polyarcrylamide gel (SDS-PAGE) usingBis-Tris 10% MES buffer according the the manufacturer AppliChem.Loading volume was 15 μl yeast culture supernatant/lane. All pGAP- andpLLP-strains were pre-tested by PCR for scFv. Only scFv-positive strainswere subsequently tested for expression of scFv protein. The table belowthe SDS PAGE gel lists the protein bands at the molecular weightpositions of LLP and scFV, the intensity of the protein band (++ strongband, + clear band, o faint band, − no band) as well as the Optionaccording to FIG. 1 to which this yeast clone belongs (Option 1: onlyGOI is expressed, no LLP; Option 2: GOI and LLP are expressed)

-   M: Protein Marker VI prestained (AppliChem)-   Lane 1 scFv control strain that did not grow very well in deep well    plates-   Lanes 2-9: strain YJK_PVA_021, expressing low amounts of PVA under    control of the GAP-promoter (faint band of scFV protein can be seen    in lanes 2-9)-   Lanes 10-18: strain PP_ESBA521_010 expressing scFv protein under    control of the LLP-promoter.-   Lanes 10 to 14: strong protein band at the molecular weight of scFv    (see ←FscFv) (“←” indicates the protein band corresponding to the    scFv protein) no protein band at the molecular weight of LLP (see    ←FLLP), example of Option 1 (only expression of GOI)-   Lanes 15 and 16: strong protein band at molecular weight of LLP (see    ←FLLP) (“←” indicates the protein band corresponding to the LLP    protein) and clear band of scFv at molecular weight of scFv (see    ←FscFv), example of Option 2 (parallel expression of GOI and of LLP)-   Lane 17: clear protein band at molecular weight of LLP (see ←FLLP)    and strong protein band of scFv at molecular weight of scFv (see    ←FscFv), example of Option 2-   Lane 18: no protein band at molecular weight of LLP (see ←FLLP) and    strong protein band of scFv at molecular weight of scFv (see    ←FscFv), example of Option 1

The high molecular weight protein band in the gel at the position ofabout the molecular weight marker 125 kDa represents a dimer of theLLP-protein. The identity of the LLP-protein was proven by the followingexperiments and evidences. First the LLP-band was cut out of the gel andthe C-terminal amino acid sequence was determined by peptide sequencingusing standard methods know in the art. Furthermore the LLP-gel band wasanalyzed by mass spectrometry using standard methods known in the art.Both methods proved that this protein band in the gel indeed isLLP-protein. Furthermore LLP was treated with an enzyme removingglycostructures from proteins, namely PNGase F. Treatment of LLP withPNGase F lowered the molecular weight of the LLP-dimer from about 125kDa to about 110 kDa. Furthermore it is known from the literature (IUBMBLive, 2009, 61:252-60), that lectins can form very stable multimers suchas dimers. Therefore we conclude the high molecular LLP-protein band inour SDS-PAGE gels is a stable LLP-protein dimer.

FIG. 5 A: SDS PAGE Analysis

A codon optimized DNA sequence (SEQ ID NO: 17) encoding the modelprotein hGH was inserted into a plasmid with the GAP-promoter (pGAP) andone plasmid with the LLP-promoter (pLLP). The pGAP plasmid wastransformed in NRRL Y-11430 and the pLLP plasmid in SSS1. For pGAP, 16transformants were randomly picked and subjected to expression studiesat microtiter plate scale (25° C., 350 rpm, 70 h). For pLLP, 28transformants were randomly picked and subjected to expression studiesat microtiter plate scale (25° C., 350 rpm, 70 h). The OD at 600 nm wascomparable at harvest (OD was determined at harvest to check for biomassvariability). Subsequently, 15 μL of the samples (supernatant mixed withNuPAGE® LDS sample buffer incl. 2-mercaptoethanol) were loaded onto thegel.

-   M: Protein Marker VI 10-245 from AppliChem.-   pGAP: a protein band corresponding to hGH was detected by visual    inspection of SDS-PAGE gels in 0/16 randomly picked clones (without    prior PCR screening)-   pLLP: a protein band corresponding to hGH was detected by visual    inspection of SDS-PAGE gels in 8/28 randomly picked clones (without    prior PCR screening)

This result indicates that the pLLP system is superior to the pGAPsystem.

FIG. 5 B: hGH Western Blot

15, 20 or 30 μl supernatant samples of deep well plate cultures ofPichia pastoris cells transformed as described under FIG. 5 A weredirectly loaded on SDS-PAGE gels (Novex NuPage 4-12% Bis-Tris Gels fromInvitrogen with MES-running buffer). The proteins were then transferredto a PVDF membrane. After transfer and blocking, the membrane wasincubated with a solution containing anti-hGH antibody (Zymed Cat.Nr.18-0090, dilution: 1:1000) for 2 h. After washing the membrane, asecondary antibody coupled with alkaline phosphatase (anti-rabbit IgGalkaline-phosphatase conjugate, Sigma Cat. No. A3687, dilution:1:16.000) was added for 1 h. After washing the membrane, NBT/BCIP(nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphatetoluidine salt) was added to detect hGH.

-   M: Protein Marker VI;-   NRRL Y-11430 Control: wild type strain without hGH;-   SSS1 Control: supersecretor strain without hGH;-   pGAP: expression of hGH under control of GAP-promotor;-   pLLP: expression of hGH under control of the LLP-promoter

FIG. 6: SDS PAGE Analysis

Lane 6 contains a sample of SSS1-cells not transfected with a pLLPconstruct (negative control).

SDS-PAGE analysis. A codon optimized DNA sequence encoding the modelprotein HSA was inserted into on plasmid with the GAP-promoter (pGAP)and one plasmid with the LLP-promoter (pLLP). The pGAP plasmid wastransformed in NRRL Y-11430 and the pLLP plasmid in SSS1. Colony PCR wasapplied to screen for transformants which were then subjected toexpression studies at microtiter plate scale (25° C., 350 rpm, 70 h).The OD at 600 nm was comparable at harvest (OD was determined at harvestto check for biomass variability). Subsequently, 15 μL of the samples(supernatant mixed with NuPAGE® LDS sample buffer incl.2-mercaptoethanol) were loaded onto the gel.

-   M: Protein Marker VI 10-245 from AppliChem.-   pGAP: a faint protein band corresponding to HSA was detected by    visual inspection of SDS-PAGE gels in 2/5 selected clones (lanes 3    and 5) (with prior PCR screening)-   pLLP: a protein band corresponding to HSA was detected by visual    inspection of SDS-PAGE gels in 4/4 selected clones (with prior PCR    screening)

This result indicates that the pLLP system is superior to the pGAPsystem.

FIG. 7 Comparison of pGAP expression system with pLLP expression system

This result indicates that the pLLP system is superior to the pGAPsystem.

7A:

Pichia pastoris strains transformed with HSA either under control of theGAP promoter (pGAP=P. pastoris glyceraldehyde-3-phosphate dehydrogenasepromotor) or under control of the LLP promoter (pLLP=P. pastoris LectinLike Protein promotor) were cultured in a 5 liter fermenter for 144 h at25° C., pH 6.0 and 30% oxygen, using glycerol in batch growth phase andusing constant glucose feed in main growth stage. Culture supernatantwas analyzed for HSA employing a Human Serum Albumin ELISA (EnzymeLinked Immuno Sorbent Assay) kit from Cygnus Technologies (Cat. Nr.F055) according to the manufacturer's instructions. Mixtures oftransformed yeast strains (pGAP) and mixtures of transformed yeaststrains (pLLP) were tested in order to account for expressiondifferences in individual strains, e.g. to determine the averageexpression rate of pGAP- and of pLLP-strains.

7B:

PVA enzyme assay. A codon optimized DNA sequence encoding PVA (Pleurotusostreatus—Penicillin V Amidase) was inserted into a plasmid with theGAP-promoter (pGAP) and into a plasmid with the LLP-promoter (pLLP). ThepGAP plasmid was transformed in NRRL Y-11430 and the pLLP plasmid inSSS1 yeast cells. For each plasmid, 16 transformants were randomlypicked and subjected to expression studies at microtiter plate scale(25° C., 350 rpm, 70 h). The PVA titer (g/kg phenoxy acetic acid) and ODat 600 nm were determined at harvest and used for normalization (g/kgphenoxy normalized). The diagram shows the mean value±SEM (standarderror of the mean) of PVA activity as measured byPVA-mediated-conversion of phenoxymethylpenicillin into phenoxy aceticacid g/kg. The highest individual value in each group is labeled with anasterix (*). For expression purpose one would usually choose thosestrains which do express the highest amount of PVA-activity (labeledwith * in the diagram). In this case the best strain expressing PVAunder control of the pLLP expressed about 2.9-fold more PVA-activity ascompared to the best pGAP strain (pGAP: n=13, max. value was 0.17 g/kg,pLLP: n=15, max. value was 0.49 g/kg). The pGAP vector from DNA2.0 Inc.and the pGAP vector from Sandoz are almost identical. All Vectorelements such as promoters, terminators, resistance-marker, pUC ori,etc. are the same in both vectors. Only minor differences regarding theused restriction enzyme sites and short nucleotide sequences whichconnect some of these elements within the vectors and are slightlydifferent between both vectors.

FIG. 8: Expression of gene of interest (GOI) in fermentor yeast cultures

8A

A codon optimized DNA sequence encoding the enzyme PVA (Penicillin VAmidase) was inserted into a plasmid under the control of theGAP-promoter (pGAP). The pGAP plasmid was transformed in NRRL Y-11430yeast cells (Pichia pastoris), and cells were grown in a fermentor understandard conditions using standard media and culturing conditions asknow in the art. The pGAP vector integrated randomly into the SSN6-gene,thereby activating the LLP-promoter. The supernatant of the fermentationculture was subsequently treated with the enzyme PNGase F (PeptideN-Glycosidase F; cleaves asparagine-linked high mannose as well ashybrid complex oligosaccharides from glycoproteins; New England Biolabs,Catalogue Number P0704S). Samples of the untreated cell culturesupernatant (lane 1) and of PNGase F-treated cell culture supernatant(lane 2) were separated in SDS-PAGE. Lane 1 shows the glycosylatedLLP-dimer as a very strong band at about 125 kDa, whereas the PVA-bandis somewhat diffuse at around 72 kDa, probably because individualmolecules of PVA are glycosylated to a different extend. Lane 3 shows atabout 36 kDa the protein band of the added PNGase F, at about 110 kDathe very strong band of the deglycosylated LLP-dimer (←LLP de-glyc.) andat about 52 kDa a now very clear, distinct band representingdeglycosylated PVA (←PVA de-glyc.).

8B

A codon optimized DNA sequence encoding a single chain antibody fragment(scFv), in this case the scFv named DLX521, was inserted into a plasmidunder control of the LLP-promoter (pLLP). The pLLP plasmid containing aGeneticin resistance was randomly transformed into YJK_PVA_021 (thealready PVA-transformed yeast cells of FIG. 8 A), and cells were grownin a fermentor under standard conditions using standard media andculturing conditions as know in the art. Supernatant of the fermentationwas diluted 20-fold and 15 μl were loaded onto SDS-PAGE in the presenceof 2-mercaptoethanol. A very strong protein band at the molecular weightposition of the scFv is visible in the gel in lane 1. In addition aminor band at the position of the LLP-dimer is seen at about 125 kDa anda strong protein band of PVA is seen at about 72 kDa. No PNGase wasadded so all proteins are glycosylated. 20-fold dilution of the cellculture supernatant was done in order to depict the very high expressionrate of the scFv relative to the LLP. This yeast strain is an example ofthe “Option 2” as depicted in FIG. 3 D, except that 2 GOI are expressed(PVA and DLX521). PVA is under control of the GAP promoter and randomlyintegrated by chance into the ssn6-like gene.

This is an example showing, that besides the LLP promoter also otherpromotors, such as the GAP promotor, can be used in conjunction with theexpression system of the invention.

8C

This scheme shows the expression construct used in FIG. 8 A. A codonoptimized DNA sequence encoding the enzyme PVA under control of theGAP-promoter was inserted into a plasmid (pGAP). This pGAP plasmid wastransformed in NRRL Y-11430 yeast cells (Pichia pastoris), and cellswere grown in a fermentor under standard conditions using standard mediaand culturing. OD600 measurements were used to adjust cell densities.The pGAP vector integrated randomly into the ssn6 gene, therebyactivating the LLP-promoter and resulting in an expression of LLPprotein.

This expression construct is a proof-of-principle, again showing thatinterrupting the ssn6 gene results in an improved expression of the genebeing under control of the LLP promoter, in this case in an improvedexpression of the LLP protein, in addition to the expression of the PVAprotein.

FIG. 9:

A:

Partial alignment of 39 different protein sequences showing sequencehomology to P. pastoris SSN6-like protein (CCA36593.1) (SEQ ID NO: 8)

B:

P. pastoris SSN6-like consensus sequence 1 (SEQ ID NO: 63)

C:

P. pastoris SSN6-like consensus sequence 2 (SEQ ID NO: 64)

FIG. 10: Expression vector pLLP

This figure shows the expression vector pLLP without inserted GOI (FIG.10 A), and its full length sequence (FIG. 10 B) (SEQ ID NO: 65).

Expression vector (without inserted GOI): The gene of interest (GOI) canbe inserted into this vector using the restriction endonuclease cleavagesites NotI and/or EcoRV, resulting in the GOI being under control of theLLP promoter and the ADH terminator. In order to insert the expressionvector into the yeast genome, the vector can be linearized by cleavingwith the restriction endonuclease AvrII, which restriction site islocated between the LLP promoter and the LLP terminator sequence. Theresulting linearized vector contains at its 5′-end the LLP promoter andat its 3′-end the LLP terminator. If inserted into the yeast genome byhomologous recombination via the LLP promoter- and the LLPterminator-sequences, this homologous recombination removes the nativeLLP-coding sequence from the yeast genome. At the same time the GOIunder control of the LLP promoter and the ADH terminator as well as aZeocin expression cassette and all other parts of the pLLP vector areinserted into the yeast genome.

FIG. 11: Expression vector pGAP with DLX521 as GOI This figure shows theexpression vector pGAP with DLX521 as GOI (FIG. 11 A), and its fulllength sequence (FIG. 11 B) (SEQ ID NO: 66).

Linearization of the vector has been carried out with SwaI.

FIG. 12: Expression of HSA using LLP-signal sequence

This figure shows the expression of human serum albumin (HSA) utilizingthe secretion signal sequence of LLP. The LLP-signal sequence was fusedN-terminally to the coding sequence of HSA (llps-HSA), transformed intoyeast cells. 10 randomly picked clones were grown in deep well platesand the supernatant checked for HSA expression (FIG. 12 A). One clonewas expressed also in a 1 liter fermenter for 48, 72 and 96 hours (FIG.12 B). Expression was shown by SDS-PAGE followed by Coomassie bluestaining.

FIG. 13 A-D: Analysis of the functionality of LLP-promoter fragments(Truncation analysis)

The LLP-promoter was successively shortened by cloning of PCR-generatedLLP-promoter fragments into YJK_PVA_021 yeast strain. 11 randomly pickedclones for each LLP-promoter length were picked and grown in deep wellplates and the supernatant analyzed by SDS-PAGE followed by Coomassieblue staining. The lengths of the tested LLP-promoter fragments were asfollows: Fig. A: 576 bp, Fig. B: 512 bp, Fig. C: 472 bp, and Fig. D: 372bp. Each figure shows two representative lines, with “Option 1” denotingthat only scFv is expressed, and with “Option 2” denoting that scFv andLLP is expressed.

FIG. 14: Sequences S. cerevisae ssn6 and TUP1 (complete coding region)

This figure shows the respective complete coding region of thefollowing:

A. Ssn6 nucleic acid, of S. cerevisiae (SEQ ID NO: 135);

B: SSN6 Protein, of S. cerevisiae (SEQ ID NO: 137);

C: Tup-1 nucleic acid of S. cerevisiae (SEQ ID NO: 136);

D: Tup-1 protein of S. cerevisiae (SEQ ID NO: 138).

METHODS

1. Assessing the Amount of a Candidate Ssn6-Like Related Gene

The level of expression of a candidate ssn6-like related gene can bemeasured for example by measuring the level of mRNA of said ssn6-likerelated gene by northern blotting or by quantitative Polymerase ChainReaction (qPCR) or reverse transcriptase qPCR, or measuring the activityof the promoter of said ssn6-like related gene for example by usingluciferase reporter gene assays or by using ssn6-like relatedpromoter-green fluorescent protein (GFP) constructs, etc. All thesemethods are well known to a person skilled in the art and representroutine work. A textbook comprising protocols for routine methods is forinstance Sambrook et al., “Molecular Cloning: A Laboratory Manual”,4^(th) Edition, Cold Spring Harbor Laboratory Press, (2012), referred toherein as Sambrook et al.

2. Assessing Whether a Candidate Ssn6-Like Related Gene Resembles theSsn6-Like Gene as Defined Herein with Respect to Function, Activity andSequence

2.1 Measuring the Amount of LLP Protein Expressed in Cell Culture:

The amount of a protein (or its expression level) can be determinedaccording to any suitable method that is known to a person skilled inthe art, for instance by measuring the amount of LLP protein in thesupernatant of a cell culture by ELISA, by western blotting, or SDS PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis ofLLP protein.

In the present invention, the amount of LLP protein in the supernatanthas been measured by carrying out a qualitative SDS PAGE analysis (NovexNuPage 4-12% Bis-Tris Gels with MES running buffer, from Invitrogen).

Alternatively the level of LLP protein can be determined indirectly bymeasuring LLP-mRNA using the same methods as described above inparagraph 1 “Assessing the amount of a candidate ssn6-like relatedgene”.

2.2 Measuring the Amount of a Gene of Interest (GOI) Expressed UnderControl of the LLP-Promoter in Cell Culture

The same methods for measuring the amount of LLP-protein, as describedabove under 2.1, can also be used for measuring GOI-protein expressedunder control of a LLP-promoter according to the invention.

2.3 Measuring Activity and Function of a Protein

In order to measure the function and activity of SSN6-like protein orSSN6-like related protein, any suitable protocol that is known to askilled person can be used. Especially any method suitable to measurethe functioning of the LLP-promotor can be use to measure the functionand activity of SSN6-like protein or SSN6-like related protein, such asthe methods mentioned under 2.1 and 2.2 above.

Once an SSN6-like protein or a SSN6-like related protein has beenidentified, its LLP-promoter-suppressing activity can be inhibited byinactivating the corresponding ssn6-like gene or ssn6-like related geneby methods described elsewhere in this application. Once this blockagehas been performed the functioning (meaning the reduced amount of, orthe complete lack of) the corresponding SSN6-like protein or mRNA orSSN6-like related protein or mRNA can be measured. Suitable methods aredescribed elsewhere in this application.

PVA enzyme assay: an aliquot of the supernatant was mixed with thesubstrate Phenoxymethylpenicillin-Kalium and incubated at 22° C. Thepresence of active PVA enzyme will result in cleavage of the substratein a titer-dependent manner. The reaction was stopped after 60 min byaddition of ice-cold methanol. The amount of the cleavage productphenoxy acetic acid was determined by quantitative HPLC.

3. Comparison of Different Expression Systems

In order to compare the protein expression of different expressionsystems, any suitable method that is known to a person skilled in theart can be carried out. In the present invention, the following protocolhas been applied for this purpose:

The supernatants obtained from the pLLP expression system and from thepGAP system (same volume) were directly loaded on SDS-PAGE gels forrelative comparisons of the pLLP vs. pGAP system.

4. Identification of Regulatory DNA Sequences

There are various tools available to predict regulatory DNA sequences,reviewed for example by Wassermann et al., Nature Reviews Genetics 5,276-287. There are also tools for the prediction the total length of apromotor sequence, as well as tools predicting distinct positions withinsuch a promoter, which supposedly bind to certain transcription factors,etc. One of these online tools is available from the University ofCopenhagen, Bioinformatics Center, under http://jaspar.binf.ku.dk/. Weused the JASPAR development server, Version 5.0_ALPHA. Settings of thisonline tool were: JASPAR CORE fungi, all JASPAR matrix models werechosen (we searched for all transcription recognition sites listed forfungi in the JASPER CORE fungi database), profile score threshold wasset to 95%. Our input sequence was the LLP-promoter sequence SEQ ID NO:12. This analysis resulted in 63 predicted binding sites fortranscription factors within the tested sequence as shown in the tablebelow.

More details on transcription factor binding site prediction can befound in Nat Rev Genet. 2004:4, 276-87.

predicted Model site name Score Start End Strand sequence SKO1 14.890274 281 1 ACGTAATG ABF1 13.893 204 219 −1 CCGTAAAAA GCGATAC (SEQ IDNO: 116) CST6 13.595 271 279 1 ATGACGTAA SUM1 11.962 346 354 1 ATAATTTTTSPT23 11.708 750 757 −1 AAAATCAA SPT23 11.708 516 523 1 AAAATCAA YOX111.696 638 645 −1 TTAATTAT RFX1 11.668 39 46 −1 CGTTGCTA STE12 11.624917 923 −1 TGAAACG YHP1 11.563 499 504 1 TAATTG AFT2 11.464 69 76 −1CACACCCT TEA1 11.362 265 272 1 GCGGACAT YML08 11.251 72 80 −1 ACCCCACAC1W ARR1 11.154 684 691 −1 ATTTGAAT TOS8 11.143 380 387 1 GTGTCAAA MBP1::10.880 717 723 1 TCGCGTT SWI6 PHD1 10.721 104 113 −1 ACCTGCATCA (SEQ IDNO: 117) YPR02 10.671 73 79 −1 CCCCACA 2C YGR06 10.573 67 80 −1ACCCCACAC 7C CCTAC (SEQ ID NO: 118) ACE2 10.293 333 339 1 CCCAGCA ADR110.260 74 80 −1 ACCCCAC MIG3 10.179 73 79 −1 CCCCACA STB5 10.040 835 842−1 CGGTATTA MIG2 10.037 73 79 −1 CCCCACA MIG1 9.822 74 80 −1 ACCCCACYAP5 9.428 704 709 −1 AAGCAT YAP5 9.428 698 703 1 AAGCAT YAP5 9.428 456461 1 AAGCAT SIG1 9.387 222 226 −1 ATATA ARR1 9.314 106 113 −1 ACCTGCATMOT3 9.052 296 301 1 AAGGTA YAP5 8.787 259 264 1 AAACAT HAP2 8.684 424428 1 TTGGT HAP2 8.684 159 163 1 TTGGT SKN7 8.580 401 406 −1 GGCCAT HAP28.495 665 669 −1 TTGGC HAP2 8.495 622 626 1 TTGGC HAP2 8.495 404 408 −1TTGGC PHO2 8.463 436 441 −1 ATAATA GLN3 8.378 570 574 1 GATAA GLN3 8.3786 10 1 GATAA MBP1 8.257 716 722 −1 ACGCGAT PHO2 8.131 119 124 1 ATATTAPHO2 8.131 90 95 −1 ATATTA SKN7 8.026 403 408 1 GGCCAA ARG80 7.974 956961 −1 TGACAC ARG80 7.974 380 385 −1 TGACAC YAP5 7.933 185 190 1 AGACATFZF1 7.932 551 556 −1 CTATCA PHO2 7.806 488 493 1 TTATTA PHO2 7.806 435440 1 TTATTA PHO2 7.733 638 643 1 ATAATT PHO2 7.733 346 351 1 ATAATTPHO2 7.733 51 56 1 ATAATT PHO2 7.408 640 645 −1 TTAATT PHO2 7.402 587592 −1 ATATTT GLN3 7.272 552 556 1 GATAG GLN3 7.272 28 32 −1 GATAG GLN37.272 2 6 1 GATAG HAP2 7.252 592 596 −1 TTGGA HAP2 7.252 529 533 1 TTGGAHAP2 7.252 453 457 −1 TTGGA HAP2 7.252 196 200 −1 TTGGA

5. Transformation and Cultivation of Strains

Expression constructs were linearized by digestion with suitablerestriction enzyme and transformed in Pichia strains by electroporation.The transformants were subsequently plated on agar plates containingZeocin (final concentration: 100 mg/L) and/or Geneticin (finalconcentration: 300 mg/L).

Single colonies or glycerol stocks were subjected to expression studiesin 48-well plates using a starch/amylase based cell culture medium. OD(Optical Density) at 600 nm at harvest was around 10.

6. Testing of Promoter Activity

Promoter activity can be measured by any suitable method that is knownto a person skilled in the art. An example of such a method is the useof qPCR or reporter gene assays (e.g. Luciferase, Green FluorescentProtein (GFP) etc.), both of which are standard methods that are knownto a person skilled in the art. For example the most suitable part ofthe LLP-promoter for high level expression of a GOI can be determined bysuccessively shortened versions of the LLP-promoter sequence accordingto SEQ ID NO. 12, by inserting such shortened LLP-promoter sequencestogether with a Kozak sequence and a model protein sequence such asDLX521 (scFv), hGH, HSA, PVA, etc. and together with a signal sequencesuch as the MF-alpha pre-pro signal sequence with or without EAEArepeat, the natural signal sequence of said model protein, etc. into apLLP vector carrying a resistance-marker such as Geneticin, Zeocin, etc.and transfecting such pLLP vector into a suitable yeast cell such as forexample YJK_PVA_021-cells, SSS1-cells, NRRL Y-11430-cells, etc.Individual clones or pooled clones of such transformed yeast cells thencan be grown under standard growth conditions in deep well plated,shaker flasks, fermentors, etc. and the amount of expression of saidmodel protein being measured using methods such as SDS-PAGE, ELISA, orprotein-activity assays such as the PVA-assay described elsewhere inthis application, etc. Shortened versions of the promoter couldrepresent parts of the promoter sequence disclosed in SEQ ID NO: 12, forexample a LLP-promoter having a length of 1000, 775, 675, 605, 576, 512,472, 415, 404, 372, 305, 285, 235, 165, 100 nucleotides, etc. counted ineach case from the 3′-end of SEQ ID NO: 12.

7. Assessing Degree of Identity of Nucleotide Sequences or Amino AcidSequences

“Sequence identity” or “% identity” refers to the percentage of residuematches between at least two polypeptide or polynucleotide sequencesaligned using a standardized algorithm. Such an algorithm may insert, ina standardized and reproducible way, gaps in the sequences beingcompared in order to optimize alignment between two or more sequences,and therefore achieve a more meaningful comparison of the sequences. Forpurposes of the present invention, the sequence identity between twoamino acid or nucleotide sequences is determined using the NCBI BLASTprogram version 2.2.29 (Jan. 6, 2014) (Altschul et al., Nucleic AcidsRes. (1997) 25:3389-3402). Sequence identity of two amino acid sequencescan be determined with blastp set at the following parameters: Matrix:BLOSUM62, Word Size: 3; Expect value: 10; Gap cost: Existence=11,Extension=1; Filter=low complexity activated; Filter String: L;Compositional adjustments: Conditional compositional score matrixadjustment. For purposes of the present invention, the sequence identitybetween two nucleotide sequences is determined using the NCBI BLASTprogram version 2.2.29 (Jan. 6, 2014) with blastn set at the followingexemplary parameters: Word Size: 11; Expect value: 10; Gap costs:Existence=5, Extension=2; Filter=low complexity activated;Match/Mismatch Scores: 2,−3; Filter String: L; m.

8. Generation of the Super-Secretor Strain (SSS1)

An expression cassette encoding the enzyme PVA (Penicillin V Amidase)under the control of the GAP promoter and another expression cassettecoding for Zeocin was transformed into the P. pastoris strain NRRLY-11430 (see table below). Clones secreting high levels of active PVAwere screened by the following enzyme assay:

The cleavage of the PVA substrate penicillin V to the products 6-APA(6-Aminopenicillanic acid) and phenoxyacetic acid was determined byquantifying the phenoxyacetic acid amount by HPLC.

One clone showing high PVA titers was identified and named YJK_PVA_021.Surprisingly, this clone not only secreted high amounts of PVA but alsothe LLP protein at even higher levels. Thus, YJK_PVA_021 was furthercharacterized by whole genome sequencing done by the company IlluminaInc., San Diego, Calif., USA. After de-novo assembly of the genome doneby Illumina Inc. (see SEQ ID NOs: 67-115, showing the results of genomicsequencing), the localization of the sequence of the PVA expressionconstruct was identified to be in SEQ ID NO: 103 and the adjacentgenomic sequences were compared to the reference strain (P. Pastoris CBS7435, gi|32835130|emb|FR839629.1|) for identification of the insertionsite. A single copy of the expression cassette encoding PVA was found tobe integrated randomly at position 807,480 of chromosome 1 of thereference strain Pichia pastoris CBS 7435. This position lies within thessn6-like gene sequence resulting in disruption of the ssn6-like geneleading to a C-terminal truncated protein (see FIG. 2 B, see SEQ ID NO:9) and to a 3′-truncated coding region of the ssn6-like gene,respectively (for details see FIG. 1 S, see SEQ ID NO: 23. No furtherobvious deviations from the reference sequence were found. Thus, thissingle random integration event resulted in high level secretion of LLP.The PVA expression cassette was removed from YJK_PVA_021 resulting inclone SSS1 (see also FIG. 3).

The vector pGAPk was PCR-amplified (linearized by PCR) using oligo2395(TCCTCGTCCAATCAGGTAG; SEQ ID NO: 119) and oligo2398(AGTGGTACCTGCAGCTAAG; SEQ ID NO: 120) and the PCR-product wastransformed into yeast strain YJK_PVA_021. Homologous recombinationreplaced the pGAP-PVA expression vector containing the Zeocin-resistancemarker with the empty vector sequence of pGAPk (empty expressioncassette and Geneticin-resistance marker). Clones withGeneticin-resistance and without PVA activity were screened. PVA wasmeasured as indicated above. One strain which does not express PVA butexpresses high amounts of LLP was denoted SSS1 and used for subsequentexpression studies of different GOIs.

9. Characterization of the LLP Signal Sequence

The secreted LLP protein was N-terminally sequenced by Edman degradationto identify the LLP signal sequence cleavage site (Seq. ID NO. 3, FIG. 1C). In order to proof the general functionality of the LLP signalsequence, resulting in the secretion of heterologous proteins fused tothe LLP signal sequence, the following experiments were performed: TheLLP signal sequence was fused to the 3′ end of HSA coding sequencereplacing the native HSA-signal sequence resulting in the sequenceaccording to SEQ. ID NO: 121, (FIG. 1 T) and cloned into the pLLPplasmid via EcoRV and NotI restriction enzyme sites. The HSA codingsequence was codon optimized (done by DNA2.0 Inc., Menelo Park, USA) anda silent point mutation was inserted into the LLP-signal sequence atposition 45 exchanging G for a C, which does not change the amino acidsequence of the LLP-signal peptide, but which deletes the restrictionenzyme site PstI from the LLP-signal sequence. The resulting plasmid wastransformed into SSS1 yeast strain. 10 clones were randomly picked andsubjected to cultivation in deep well plates. 15-20 μl/lane supernatantof deep well plate cultures were directly loaded on SDS-PAGE gels (NovexNuPage 4-12% Bis-Tris Gels from Invitrogen with MES-running buffer).FIG. 12 A shows the results of this experiment: Lanes 3 and 7 representclones bearing no HSA insert, lanes 2, 4, 8 and 10 represent clonesaccording to option 1 (only HSA expressed), and lanes 1, 5, 6 and 9represent clones according to option 2 (HSA expressed in parallel toLLP). FIG. 12 B shows the result of a 1 liter fermenter expression usingthe same clone used in lane 2 of the gel in FIG. 12 A. A sample from thefermenter supernatant was collected after 48, 72 and 96 hours and 15 μlof each sample subjected to SDS-PAGE, followed by Coomassie bluestaining using the “SimplyBlue SafeStain” system from Life Technologiesaccording to the manufacturer's instruction.

10. Characterization of the Ssn6-Like/LLP-Promoter Expression System

The table below shows the basic characteristics of the expressionconstructs/vectors and the yeast cells used in order to evaluate thefunctioning of the expression system for various classes ofproteins/genes of interest (GOIs), namely hormones, antibodies, enzymesand structural proteins. The expression results of GOI depicted in thefigures were generated with the expression constructs/vectors/hoststrain combinations listed in the table below. Comparison of theexpression of a GOI using a standard GAP-promotor or the LLP-promoterwas done using SDS-PAGE, ELISA and/or enzymatic activity assays, asdescribed in the figures.

GOI Codon inserted in Plasmid Resist- Protein Protein usage Signalvector (linearized ance Host name class for GOI sequence between with)marker strain Comments hGH Hormone codon S. EcoRV and pLLP Zeocin SSS1pLLP from optimized cerevisiae Notl (Avrll) Sandoz for Pichia MF-alphaEcoRI and pGAP Zeocin NRRL pGAP from pastoris (without Notl (Swal) Y-DNA2.0 EAEA 11430 (pJ905) repeat) DLX521 Antibody codon S. EcoRV andpLLP Zeocin SSS1 pLLP from (fragment optimized cerevisiae Notl (Avrll)Sandoz scFv) for Pichia MF-alpha EcoRI and pGAP Zeocin NRRL pGAP frompastoris (without Notl (Swal) Y- DNA2.0 EAEA 11430 (pJ905) repeat) EcoRVand pLLP Geneticin YJK_P pLLP from Notl (Avrll) VA_021 Sandoz PVA Enzymecodon S. EcoRV and pLLP Zeocin SSS1 pLLP from optimized cerevisiae Notl(Avrll) Sandoz for Pichia MF-alpha EcoRI and pGAP Zeocin NRRLY- pGAPfrom pastoris (with Notl (Bglll* 11430 Sandoz EAEA repeat) HSAStructural codon Human EcoRV and pLLP Zeocin SSS1 pLLP from proteinoptimized HSA pre- Notl (Avrll) Sandoz for Pichia pro signal EcoRI andpGAP Zeocin NRRLY- pGAP from pastoris sequence Notl I (Swal) 11430DNA2.0 (pJ905) Pichia EcoRV and pLLP Zeocin SSS1 pLLP from pastoris Notl(Avrll) Sandoz LLP- signal sequence**, *** *This plasmid was alsolinearized by PCR as described elsewhere in this application (seeparagraph 8. ″Generation of the super secretor strain (SSS1)″). **HSAcoding sequence was independently codon optimized resulting in identicalamino acid sequences but in slightly different nucleotide sequences ascompared to the HSA-sequence one row above ***P. pastoris signalsequence contains a silent point mutation at position 45 changing G to Cin order to delete a restriction enzyme site

11. Identification of Ssn6-Like Consensus Sequences

The ssn6-like sequence of Pichia pastoris strain NRRL Y-11430 was usedfor a BLAST-search of similar sequences. The BLAST was performed onhttp://blast.ncbi.nlm.nih.gov/. BLAST parameters were “automaticallyadjust parameters for short input sequences”, Expect threshold: 10, Wordsize: 3, Max matches in a querry range: 0, Matrix: BLOSUM62, Gap Costs:Existence: 11 Extension:1, Compositional adjustments: “Conditionalcompositional score matrix adjustment. The top 39 sequences originatefrom the following organisms (also see FIG. 9A): Komagataella pastorisCBS 7435 (Synonym/other names: Pichia pastoris, Pichia pastoris CBS7435), Komagataella pastoris GS115 (Synonym/other names: Pichiapastoris, Pichia pastoris GS115), Scheffersomyces stipitis CBS 6054(Synonym/other names: Pichia stipitis, Pichia stipitis CBS 6054),Millerozyma farinosa CBS 7064 (other name: Pichia farinosa CBS 7064),Candida parapsilosis, Candida orthopsilosis Co 90-125, Debaryomyceshansenii CBS767, Spathaspora passalidarum NRRL Y-27907, Candidaalbicans, Candida albicans SC5314, Candida maltosa Xu316, Candidatropicalis MYA-3404 (other name: Candida tropicalis T1), Lodderomyceselongisporus NRRL YB-4239 (other name: Saccharomyces elongisporus),Clavispora lusitaniae ATCC 4272 (genebank anamorph: Candida lusitaniaeATCC 42720), Meyerozyma guilliermondii ATCC 6260 (genebank anamorph:Pichia guilliermondii ATCC 6260), Wickerhamomyces ciferrii, Ogataeaparapolymorpha DL-1 (synonym and other names: Hansenula polymorpha,Hansenula polymorpha DL-1, Ogataea angusta DL-1, Ogataea parapolymorphaATCC 26012, Ogataea parapolymorpha DL-1, Pichia angusta DL-1),Cyberlindnera fabianii (synonyms and other names: Hansenula fabianii,Pichia fabianii, . . . ), Kuraishia capsulata CBS 1993, Dictyosteliumdiscoideum AX4 (belongs to social amoebae), Tetrapisispora phaffii CBS4417 (synonym: Fabospora phaffii, Dictyostelium purpureum (belongs tosocial amoebae), Pseudozyma flocculosa PF-1, Malassezia globosa CBS7966, Botryobasidium botryosum FD-172 SS1 (basidiomycete), Naumovozymadairenensis CBS 421 (synonyme: Saccharomyces dairenensis),Tetrapisispora blattae CBS 6284, Mucor circinelloides f. circinelloides1006PhL (Early diverging fungal lineage), Malassezia sympodialis ATCC42132, Kazachstania naganishii CBS 8797 (Saccharomyces naganishii),Saccharomyces cerevisiae YJM789, Saccharomyces cerevisiae FostersB,Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Ustilagohordei (Corn smut fungus, basidiomycete), Meyerozyma guilliermondii ATCC6260 (synonym/other names: Candida guilliermondii, Pichia guilliermondiiATCC 6260), Ustilago maydis 521, (Corn smut fungus, basidiomycete).

The top 39 identified amino acid sequences were aligned to thessn6-like-amino acid sequence (=identical to CCA36593.1) revealingsignificant similarities (see FIGS. 9 B and 9 C). The alignment was doneusing the online tool Clustal Omega provided by the EMBL-EBI(http://www.ebi.ac.uk/Tools/msa/clustalo/ which is described by Sieverset al., Molecular Systems Biology 7, article number: 539, Valentin etal., Nucleic acids research, 2010, 38, Suppl. W695-9, and by McWilliamet al., Nucleic acids research, 2013, July; 41 (Web Server issue):W597-600. Clustal Omega uses the HHaliqn algorithm and its defaultsettings as its core alignment engine. The algorithm is described inSoding, J. (2005) ‘Protein homology detection by HMM-HMM comparison’.Bioinformatics 21, 951-960. The default transition matrix is Gonnet, gapopening penalty is 6 bits, gap extension is 1 bit. The symbols used forthe consensus sequence at the bottom of the alignment are as follows. An“*” (asterisk) indicates amino acid positions which have a single, fullyconserved residue across all 40 aligned sequences. A “:” (colon)indicates conservation between groups of amino acids of strongly similarproperties—scoring>0.5 in the Gonnet PAM 250 matrix. A “.” (period)indicates conservation between groups of amino acids of weakly similarproperties—scoring ≥0.5 in the Gonnet PAM 250 matrix. SequenceCCA36593.1 corresponds to the Pichia pastoris SSN6 sequence identifiedin this application. FIG. 9 A shows only that part of the alignment withthe highest similarity between all 40 sequences. FIG. 9 B shows theconsensus sequence corresponding to Pichia pastoris SSN6-like(CCA36593.1) amino acids 352 to 372. FIG. 9 C shows the consensussequence corresponding to Pichia pastoris SSN6-like (CCA36593.1) aminoacids 394 to 417.

Amino acids which are identical in all 40 sequences are written in whitewith black background-labelling. The consensus sequence shows eitherindividual amino acids which are identical in all 40 sequences or showsa group of amino acids in brackets “( )” and each of the amino acids insuch a set of brackets can be chosen alternatively for that position inthe consensus sequence. A “-” (dash) indicates that the amino acid atthis position may also be omitted from the consensus sequence, which isfor example one possibility for positions 406 and 407 of the consensussequence depicted in FIG. 9 C. For example the starting amino acid atposition 352 of the consensus sequence in FIG. 9 B is “W”, meaning thatthe consensus sequence at this position contains a Tryptophan, position353 of the consensus sequence in FIG. 9 B is written as “(CGL)” meaningthat at this position of the consensus sequence can either be locatedCysteine, Glycine or Leucine, position 354 is labelled “(SLTA)” meaningthat at this position there can either be located Serine, Leucine,Threonine or Alanine, etc. The same nomenclature is used for the secondconsensus sequence depicted in FIG. 9 C. Consensus sequences can beshorter or longer as the two exemplary consensus sequences shown inFIGS. 9 B and 9 C. Consensus sequences can be deduced from the sequencealignment of FIG. 9A or can be deduced from other parts of the sequencealignment prepared from the 40 above mentioned sequences using thesequence alignment method as described above. Preferably the consensussequence contains at least 24 amino acids, preferably 23, 22, 21, 20,19, 18, 17 amino acids, more preferably at least 16, 15, 14, 13, 12, 11,or 10 amino acids, most preferably at least 9, 8, 7, 6, 5 or 4 aminoacids. Preferably a SSN6-like protein contains at least one or twoconsensus sequences, more preferably both consensus sequences shown inFIG. 9 B and FIG. 9 C, more preferably at least one consensus sequence,most preferably a consensus sequence selected from the sequences shownin FIGS. 9 B and 9 C.

12. Characterization of the Functionality of the LLP-Promoter

The plasmid pLLPk containing DLX521 with MFalpha signal sequence wasused as PCR-template in combination with the following PCR-primers togenerate shortened/truncated versions of the LLP-promoter (A-fragment).

Used Reverse Primer:

Sequence SEQ Primer  (5′ to ID name 3′ end) Δ-fragment NO.: 2892TGTCGAAC used for all  122 CACCACTA see FIG. 13 A C to FIG. 13 Dfragments

Used Forward Primer:

Sequence Length of SEQ Primer (5′ to 3′ Promoter ID name end) Δ-fragmentfragment NO.: Yo_218 TATACCTAGG Δ29 576 123 TGGTGGAACT TTATTATTCT TTCYo_219 TATACCTAGG Δ93 512 124 TATTAGCTGG TAATTGAGCG Yo_220 TATACCTAGGΔ133 472 125 TTGGAGGGT ATGGTCAGAG Yo_221 TATACCTAGG Δ201 404 126TTTCATTCCA TCTTGCCATC Yo_222 TATACCTAGG Δ233 372 127 CTTACATCAATAATTAAAAC Yo_223 TATACCTAGG Δ300 305 128 GCAAGCATAT GCTTAAAAGG

The resulting PCR products were ligated in via SpeI and AvrIIrestriction enzyme sites into the vector pLLPk_containing DLX521. Thecorrect sequences of the resulting plasmids were confirmed by DNAsequencing. The plasmids were linearized with AvrII and transformed instrain YJK_PVA_021. 11 clones per plasmid were randomly picked andsubjected to cultivations in deep well plates using a synthetic medium.At harvest, 15-30 μl supernatant samples were directly loaded onSOS-PAGE gels (Novex NuPage 4-12% Bis-Tris Gels from Invitrogen withMES-running buffer) to analyze expression of scFv by staining the gels(Simply Blue Safe Stain). The 00600, meaning the yeast cell numbers perml culture medium at harvest, was comparable for all clones analyzed.

M=Protein Marker VI.

Results are shown in FIG. 13 A to FIG. 13 D.

13. Examples of Ssn6-Like Mutants

The yeast strain contains the modified ssn6-like gene coding for aminoacids 1 to 367 and in addition seven amino acids (EWYLQLR; SEQ ID NO:139) originating from the vector inserted into the ssn6-like gene inorder to disrupt the ssn6-like gene. Alternatively, modified versions ofssn6-like might contain a modified ssn6-like gene coding for thefollowing regions of amino acids of SSN6-like protein, to the effectthat the modified SSN6-like protein is not able to exert its wildtypefunction and/or wildtype activity:

Modified versions of SSN6-like protein comprising amino acids 1 to 44, 1to 77, 1 to 100, 1 to 122, 1 to 155, 1 to 189, 1 to 235, 1 to 275, 1 to315, 1 to 348, 1 to 400, 1 to 450, 1 to 500, 1 to 550, 1 to 600, 1 to650, 1 to 367, 1 to 400, 1 to 450, 1 to 500, 1 to 550, 1 to 600, 1 to650, and/or 1 to 700 of SSN6-like protein according to SEQ ID NO. 9.

In another alternative version the modified versions of ssn6-like mightcontain the region of the ssn6-like gene coding for blocks of SSN6-likeamino acids according to SEQ ID NO. 9, namely amino acids 1 to 44, 45 to77, 78 to 100, 101 to 122, 123 to 155, 156 to 189, 190 to 235, 236 to275, 276 to 315, 316 to 348, 348 to 367, 368 to 400, 401 to 450, 451 to500, 501 to 550, 551 to 600, 601 to 650, 651 to 700, and/or 701 to 736.Each of these blocks of amino acids might be combined with one or moreother blocks of amino acids, preferably in the same order as they occurin SEQ ID NO. 9, wherein none, one or more amino acid block(s) is/arelacking in between two amino acid blocks.

1-27. (canceled)
 28. A polynucleotide sequence comprising a modifiedssn6-like gene, wherein either (A) or (B) applies: (A): the ssn6-likegene is defined as comprising SEQ ID NO: 1, the nucleotide sequence ofthe ssn6-like gene is modified by introduction of a point mutation, or apartial deletion, wherein, if said polynucleotide sequence is (a)introduced into a suitable expression system, and tried to be expressed,essentially no SSN6-like protein is expressed, or (b) expressed in asuitable expression system, SSN6-like protein is expressed that does notexert its wildtype function and/or wildtype activity, and/or (c)expressed in a suitable expression system, an amount of SSN6-likeprotein is expressed that differs from the amount of wildtype SSN6-likeprotein; (B): the polynucleotide sequence comprises SEQ ID NO:
 7. 29.The polynucleotide sequence according to claim 28, wherein thepolynucleotide comprises modifications of SEQ ID NO:
 1. 30. A Thepolynucleotide according to claim 28, wherein the polynucleotide is avector.
 31. An expression vector comprising a promoter, wherein saidpromoter is characterized in that it is repressed in the presence ofSSN6-like protein if said expression vector is introduced into asuitable expression system, wherein the ssn6-like protein is defined ascomprising the amino acid sequence as depicted in SEQ ID NO: 8, andwherein said promoter is an LLP promoter comprising SEQ ID NO: 133 ormodified versions thereof, said modified versions being characterized inthat they still exhibit the promoter function, wherein an LLP protein,which is encoded by the nucleotide sequence depicted in SEQ ID NO: 16,is not encoded by the polynucleotide sequence of this expression vector.32-33. (canceled)
 34. The expression vector of claim 31, which furthercomprises one or more gene(s) of interest, wherein this one or moregene(s) of interest is under control of said LLP promoter. 35.(canceled)
 36. A host cell, comprising the vector according to claim 30.37-45. (canceled)
 46. The polynucleotide sequence according to claim 28,wherein, if said polynucleotide sequence is expressed in a suitableexpression system, or is introduced in a suitable expression system andtried to be expressed, the amount of SSN6-like protein is reduced, orthere is no SSN6-like protein at all, compared to the amount ofSSN6-like protein that is expressed when the corresponding wildtypepolynucleotide sequence is expressed under comparable or the sameconditions.
 47. The polynucleotide sequence according to claim 28,comprising SEQ ID NO:
 7. 48. The expression vector of claim 34, whereinthe one or more gene(s) of interest is (are) selected from the groupconsisting of genes encoding enzymes, antibodies or fragments thereof,hormones, structural proteins, and protein-antigens being present invaccines.
 49. A host cell, comprising an expression vector according toclaim 31.